WO2023135978A1 - Drug for treating aftereffects of novel coronavirus infection - Google Patents

Drug for treating aftereffects of novel coronavirus infection Download PDF

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WO2023135978A1
WO2023135978A1 PCT/JP2022/044828 JP2022044828W WO2023135978A1 WO 2023135978 A1 WO2023135978 A1 WO 2023135978A1 JP 2022044828 W JP2022044828 W JP 2022044828W WO 2023135978 A1 WO2023135978 A1 WO 2023135978A1
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sequelae
novel coronavirus
covid
protein
aftereffects
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PCT/JP2022/044828
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French (fr)
Japanese (ja)
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一博 近藤
直美 岡
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学校法人慈恵大学
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/02—Nasal agents, e.g. decongestants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/24—Antidepressants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C07K14/08—RNA viruses
    • C07K14/165—Coronaviridae, e.g. avian infectious bronchitis virus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage

Definitions

  • the present invention relates to a therapeutic drug for the aftereffects of a new coronavirus infection, a method for screening a therapeutic drug for the aftereffects of a new coronavirus infection, and a method for producing a model animal for the aftereffects of a new coronavirus infection.
  • the acute respiratory disease COVID-19 (so-called novel coronavirus infection) caused by the SARS-CoV-2 virus is a serious infectious disease that has caused many infected people around the world.
  • COVID-19 is known to cause death in patients by causing serious illness in the acute phase, and to frequently cause aftereffects (hereinafter referred to as "new coronavirus infection sequelae") during the recovery period. .
  • new coronavirus infection sequelae aftereffects of COVID-19
  • fatigue, depression, and olfactory disturbance are said to be particularly common.
  • the aftereffects of COVID-19 can occur even in patients with less severe symptoms in the acute phase, it is highly likely that they will occur even if the severity of the symptoms is suppressed by the vaccine.
  • the aftereffects of the new coronavirus infection are the aftereffects of viral infection, so when the aftereffects occur, the SARS-CoV-2 virus, the causative virus, does not proliferate. Therefore, even if a drug capable of suppressing virus proliferation is administered after the onset of the aftereffects of the new coronavirus infection, it cannot be expected to be effective against the aftereffects. Therefore, it is thought that therapeutic agents for the aftereffects of novel coronavirus infection need to repair tissue damage caused by SARS-CoV-2 virus infection.
  • An object of one aspect of the present invention is to provide a therapeutic drug for the aftereffects of the novel coronavirus infection.
  • the present inventors found for the first time that the S1 region of the Spike protein of the SARS-CoV-2 virus is the causative protein of the aftereffects of the new coronavirus infection.
  • the present inventors found that acetylcholine in the brain is reduced in the aftereffects of the new coronavirus infection, and receptor activation
  • drugs can treat or prevent the aftereffects of COVID-19.
  • one aspect of the present invention is a drug for treating sequelae of novel coronavirus infection, which contains an acetylcholine receptor agonist as an active ingredient.
  • one aspect of the present invention is an administration step of administering a test substance to a novel coronavirus infection sequelae model animal in which SARS-CoV-2 S1 protein is expressed in a non-human mammal; and an evaluation step of evaluating changes in symptoms related to the novel coronavirus before and after administration of the test substance in the model animal.
  • one aspect of the present invention is a novel coronavirus infection sequelae model animal, comprising an expression step of expressing the SARS-CoV-2 S1 protein in a non-human mammal using a SARS-CoV-2 S1 protein expression vector. manufacturing method.
  • FIG. 3 shows the results of Examples, and shows the results of measuring intracellular calcium concentrations in 3T3 cells and A549 cells transformed with an S1 protein expression plasmid or a control plasmid.
  • FIG. 3 shows the results of Examples and shows the results of measuring intracellular calcium concentration in 3T3 cells and A549 cells infected with S1 protein-expressing adenovirus or control adenovirus.
  • FIG. 10 shows the results of the Examples and shows the results of the 10% weighted forced swimming test in control mice and S1 protein-expressing mice (S1 mice).
  • FIG. 4 shows the results of the Examples and shows the results of the tail suspension test in control and S1 mice.
  • FIG. 10 shows the results of Examples, and shows the results of analyzing the calbindin gene expression level in the olfactory bulb after LPS administration in control mice and S1 mice.
  • FIG. 10 shows the results of Examples, and shows the results of analyzing the calbindin gene expression level in the brain after administration of LPS in control mice and S1 mice.
  • FIG. 1 shows the results of the Examples and shows the damage of cholinergic neurons in control and S1 mice.
  • FIG. 1 shows the results of the Examples and shows the damage of cholinergic neurons in control and S1 mice.
  • FIG. 3 is a diagram for explaining the administration scheme of donepezil.
  • FIG. 2 shows the results of the Examples, showing the results of the 10% weighted forced swim test after administration of donepezil in control and S1 mice.
  • FIG. 4 shows the results of the Examples and shows the results of the tail suspension test after administration of donepezil in control and S1 mice.
  • the results of Examples are shown, and the results of analyzing the gene expression levels of interleukin 6 (IL-6), tumor necrosis factor (TNF ⁇ ), and chemokine CC motif ligand 2 (CCL2) after administration of donepezil in control mice and S1 mice.
  • IL-6 interleukin 6
  • TNF ⁇ tumor necrosis factor
  • CCL2 chemokine CC motif ligand 2
  • FIG. 10 shows the results of Examples, and shows the results of analyzing the gene expression levels of interleukin-1 beta (IL-1 ⁇ ) and interleukin-6 (IL-6) after administration of PNU282987 in control mice and S1 mice.
  • FIG. 10 shows the results of Examples, and shows the results of analyzing the gene expression level of ZFP36 after administration of PNU282987 in control mice and S1 mice.
  • a therapeutic drug for the aftereffects of novel coronavirus infection (COVID-19) according to one aspect of the present invention is a pharmaceutical composition used for treatment or prevention of COVID-19 aftereffects. , containing an acetylcholine receptor agonist as an active ingredient. This can treat or prevent the sequelae of COVID-19 in patients infected with the novel coronavirus. Therefore, it can contribute to Goal 3 of the Sustainable Development Goals (SDGs) "Good health and well-being for all”.
  • SDGs Sustainable Development Goals
  • the therapeutic drug for the aftereffects of COVID-19 according to one aspect of the present invention may be simply referred to as "therapeutic drug”.
  • treatment refers to complete cure or alleviation of symptoms of COVID-19 sequelae in a subject to whom the therapeutic agent according to one aspect of the present invention is administered (hereinafter simply referred to as “administration subject”). , including reducing the exacerbation of symptoms of COVID-19 sequelae.
  • prevention includes suppressing or delaying the onset of symptoms of COVID-19 aftereffects in a subject.
  • the therapeutic agent according to one aspect of the present invention is used for the treatment of the aftereffects of COVID-19. If the administration subject does not develop symptoms of the aftereffects of COVID-19, the therapeutic agent according to one aspect of the present invention is used for prevention of the aftereffects of COVID-19.
  • treatment and “prevention” are as described above, and in both cases the mechanism of action of the therapeutic agent according to one aspect of the present invention is the same. Therefore, “therapeutic agent” can be replaced with “prophylactic agent.”
  • 'treatment' means 'prevention' if the novel coronavirus-infected patient does not develop symptoms of COVID-19 sequelae.
  • the aftereffects of COVID-19 generally refer to symptoms in general that appear during the recovery period after infection with the new coronavirus and persist for several weeks to several months afterward, but the definition is not established.
  • the aftereffects of COVID-19 are sometimes called Long COVID.
  • "COVID-19 sequelae" in the present specification are fatigue, depressive symptoms, olfactory disorders, memory impairment, decreased concentration, decreased thinking ability, decreased cognitive function, etc., which are particularly frequent among the above symptoms. Refers to symptoms associated with nerve dysfunction.
  • COVID-19 sequelae in this specification is fatigue associated with the new coronavirus. Also, one aspect of “COVID-19 sequelae” herein is depressive symptoms associated with the novel coronavirus. In addition, one aspect of the "COVID-19 sequelae” in this specification is olfactory disturbance associated with the novel coronavirus. Also, one aspect of “COVID-19 sequelae” herein is memory impairment associated with the novel coronavirus.
  • depression symptoms associated with the new coronavirus refer to symptoms of diseases that are generally diagnosed as depression. Symptoms include depressed mood, loss of interest and pleasure, anorexia, overeating, sleep disturbance, hypersomnia, psychomotor agitation or inhibition, fatigability, feelings of worthlessness and guilt, and poor concentration. In addition to symptoms used in the diagnosis of major depression according to the DSM-5, such as slowed thinking and suicidal ideation, anxiety, memory loss, aging, pain, chronic pain, etc., are frequently seen in depressed patients. Intended to be symptomatic depression.
  • the "depressive symptoms associated with the novel coronavirus” in the present specification are not limited to major depression, and include the above depressive symptoms such as stress depression, bipolar disorder depression, and negative depression. It is a concept that includes symptoms in depression in disease and other diseases.
  • fatigue associated with the novel coronavirus is a persistent or chronic phenomenon associated with diseases of the central nervous system, such as loss of interest and pleasure, sleep disturbance, and psychomotor agitation. Or it means pathological fatigue whose main symptoms are retardation, fatigability, poor concentration and poor thinking. “Fatigue” can also be expressed as tiredness or malaise.
  • Phathological fatigue as used herein means persistent or chronic fatigue associated with the novel coronavirus infection.
  • Physiological fatigue refers to the phenomenon of temporary qualitative or quantitative decline in physical and mental work capacity seen when a healthy person is subjected to a continuous physical or mental load. are distinguished.
  • olfactory disorders associated with the new coronavirus are symptoms or diseases that cause some kind of abnormality in the sense of smell, that is, the sense of "smell”. Also known as olfactory abnormality.
  • the main symptoms include anosmia, olfactory illusion, anosmia, and hyperosmia.
  • memory impairment associated with the novel coronavirus means a state in which any or all of the four processes that make up memory - recording, retention, reproduction, and recognition - do not function properly.
  • symptoms means phenomena and conditions (abnormalities) that occur in patients due to the effects of diseases associated with the novel coronavirus, and the patients themselves can perceive that they are symptoms of the disease. It is a concept that includes subjective symptoms that are abnormal and objective symptoms that are abnormal and can be objectively confirmed to be symptoms of the disease by medical examination or examination by a doctor. In the animal model described later, changes in behavior and objective findings from examinations are collectively referred to as "symptoms.”
  • ChAT choline acetyltransferase
  • the present inventors found that by administering an acetylcholine receptor agonist, depressive symptoms and fatigue expressed in COVID-19 sequelae model animals It was found for the first time that the symptoms of In other words, the present inventors have found for the first time that the aftereffects of COVID-19 can be treated or prevented by administering an acetylcholine receptor agonist.
  • the results show that increasing the amount of acetylcholine in the brain by administering an acetylcholine receptor agonist can improve the depressive symptoms associated with the new coronavirus expressed in model animals with the aftereffects of COVID-19.
  • the result is completely opposite to the result expected as an effect of an acetylcholine receptor agonist from the conventional technical common sense.
  • depressive symptoms due to the aftereffects of COVID-19 that is, "depressive symptoms associated with the new coronavirus" develop by a different onset mechanism from depression caused by an increase in the amount of acetylcholine.
  • a therapeutic agent according to one aspect of the present invention contains an acetylcholine receptor agonist as an active ingredient.
  • acetylcholine receptor agonist refers to an indirect acetylcholine receptor agonist that increases the amount of acetylcholine in the brain by cholinesterase inhibitory action or the like, and a direct acetylcholine receptor agonist that acts by directly binding to the receptor. Both types of acetylcholine receptor agonists are contemplated.
  • the acetylcholine receptor agonist may be a peripheral acetylcholine receptor agonist that acts via parasympathetic nerves or the like, or a central acetylcholine receptor agonist that acts on receptors in the brain. It may be an agonist.
  • the aftereffects of COVID-19 are symptoms related to brain and nerve dysfunction. It is preferably a central acetylcholine receptor agonist that acts on acetylcholine receptors.
  • "Acetylcholine receptor agonists" are also referred to as cholinergics.
  • Receptors in the brain on which central acetylcholine receptor agonists act include, more specifically, the olfactory bulb, hippocampus, septal area, and/or olfactory tubercle. It is known that there are a number of locations on which is acted, and it is not limited to those exemplified here.
  • a direct-type acetylcholine receptor agonist is preferably a substance that can cross the blood-brain barrier to reach the brain and has the effect of activating the acetylcholine receptor. More specific examples of direct acetylcholine receptor agonists include acetylcholine and its precursors, and agonists of acetylcholine receptors (muscarinic receptors or nicotinic receptors).
  • the indirect acetylcholine receptor agonist is preferably a substance that can cross the blood-brain barrier to reach the brain and has an acetylcholinesterase inhibitory action.
  • Indirect acetylcholine receptor agonists more specifically, donepezil (2-[(1-benzyl-4-piperidinyl)methyl]-5,6-dimethoxyindan-1-one hydrochloride); amine (2,6-dioxo-4-phenyl-piperidine-3-carbonitrile); metrifonate (O,O-dimethyl-2,2,2-trichloro-1-hydroxyethylphosphonate ester); tacrine (1 , 2,3,4-tetrahydro-9-aminoacridine); galantamine (galantamine hydrobromide);
  • the acetylcholine receptor agonist is preferably donepezil.
  • the acetylcholine receptor agonist may be in the form of a "derivative” or a “pharmacologically acceptable salt” as long as it has the effect of increasing the amount of acetylcholine in the brain. good. That is, in the present specification, the term “acetylcholine receptor agonist” is a concept including its “derivatives” and “pharmacologically acceptable salts”.
  • the term "derivative" refers to a group of compounds produced by substituting a part of the molecule of a specific compound with another functional group or another atom.
  • other functional groups include alkyl groups, alkoxy groups, alkylthio groups, aryl groups, aryloxy groups, arylthio groups, arylalkyl groups, arylalkoxy groups, arylalkylthio groups, arylalkenyl groups, arylalkynyl groups, and allyl.
  • Examples of the other atoms include carbon atoms, hydrogen atoms, oxygen atoms, nitrogen atoms, sulfur atoms, phosphorus atoms, halogen atoms and the like.
  • prodrug that exhibits the desired activity by hydrolysis, oxidation, enzymatic reaction, or the like in vivo (under in vivo conditions).
  • the term "pharmaceutically acceptable salt” intends a salt that is physiologically acceptable for administration to a subject as a pharmaceutical, and specific examples thereof are not limited.
  • salts include alkali metal salts (potassium salts, etc.), alkaline earth metal salts (calcium salts, magnesium salts, etc.), ammonium salts, organic base salts (trimethylamine salts, triethylamine salts, pyridine salts, picoline salts, dicyclohexylamine salt, N,N'-dibenzylethylenediamine salt, etc.), organic acid salt (acetate, maleate, tartrate, methanesulfonate, benzenesulfonate, formate, toluenesulfonate, trifluoroacetate) etc.), inorganic acid salts (hydrochlorides, hydrobromides, sulfates, phosphates, etc.).
  • the acetylcholine receptor agonist may activate or inhibit receptors other than the acetylcholine receptor, but from the viewpoint of suppressing unintended side effects, etc., the acetylcholine receptor agonist is selectively used. It is preferably a (specifically) activating compound.
  • the therapeutic agent according to one aspect of the present invention may contain ingredients other than the active ingredient (acetylcholine receptor agonist) described above.
  • Ingredients other than the active ingredient may be pharmaceutically acceptable ingredients, such as buffers, pH adjusters, tonicity agents, preservatives, antioxidants, high molecular weight polymers, excipients, solvents and so on.
  • buffers examples include phosphoric acid or phosphate, boric acid or borate, citric acid or citrate, acetic acid or acetate, carbonic acid or carbonate, tartaric acid or tartrate, ⁇ -aminocaproic acid, trometamol etc.
  • phosphate examples include sodium phosphate, sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium phosphate, potassium dihydrogen phosphate, and dipotassium hydrogen phosphate.
  • borate examples include borax, sodium borate, and potassium borate.
  • the citrate include sodium citrate, disodium citrate, and trisodium citrate.
  • acetate examples of the carbonate include sodium carbonate and sodium hydrogen carbonate.
  • tartrate examples include sodium tartrate and potassium tartrate.
  • pH adjuster examples include hydrochloric acid, phosphoric acid, citric acid, acetic acid, sodium hydroxide, potassium hydroxide, and the like.
  • tonicity agents examples include ionic tonicity agents (sodium chloride, potassium chloride, calcium chloride, magnesium chloride, etc.) and nonionic tonicity agents (glycerin, propylene glycol, sorbitol, mannitol, etc.). mentioned.
  • antiseptic examples include benzalkonium chloride, benzalkonium bromide, benzethonium chloride, sorbic acid, potassium sorbate, methyl parahydroxybenzoate, propyl parahydroxybenzoate, and chlorobutanol.
  • antioxidants examples include ascorbic acid, tocopherol, dibutylhydroxytoluene, butylhydroxyanisole, sodium erythorbate, propyl gallate, and sodium sulfite.
  • high molecular weight polymers examples include methylcellulose, ethylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxyethylmethylcellulose, hydroxypropylmethylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, hydroxypropylmethylcellulose acetate succinate, hydroxypropylmethylcellulose phthalate. , carboxymethylethylcellulose, cellulose acetate phthalate, polyvinylpyrrolidone, polyvinyl alcohol, carboxyvinyl polymer, polyethylene glycol, atelocollagen, and the like.
  • excipient examples include lactose, sucrose, D-mannitol, xylitol, sorbitol, erythritol, starch, and crystalline cellulose.
  • Examples of the solvent include water, physiological saline, and alcohol.
  • the therapeutic agent according to one aspect of the present invention may contain a medicinal ingredient having the desired effect (e.g., reduction of side effects) as the other component described above, and may be used in combination with a drug having the desired effect. .
  • the amount of active ingredient in the therapeutic agent according to one aspect of the present invention is not particularly limited.
  • the amount of active ingredient may be, for example, 0.001% to 100% by weight, or 0.01% to 100% by weight, relative to the total weight of the therapeutic agent according to one aspect of the present invention.
  • the amount of ingredients other than the active ingredient in the therapeutic agent according to one aspect of the present invention is not particularly limited.
  • the amount of ingredients other than the active ingredient may be, for example, 0% to 99.999% by weight, or 0% to 99.99% by weight, relative to the total weight of the therapeutic agent according to one aspect of the present invention.
  • % may be 0 wt% to 99.9 wt%, may be 5 wt% to 99.9 wt%, or may be 10 wt% to 99.9 wt% well, it may be from 20 wt% to 99.9 wt%, it may be from 30 wt% to 99.9 wt%, it may be from 40 wt% to 99.9 wt%, it may be 50 wt% may be ⁇ 99.9 wt%, may be 60 wt% to 99.9 wt%, may be 70 wt% to 99.9 wt%, may be 80 wt% to 99.9 wt% %, or 90% to 99.9% by weight.
  • An example of a subject to whom the therapeutic agent according to one aspect of the present invention is administered includes a subject suspected of being infected with, or confirmed to be infected with, the novel coronavirus.
  • Such an administration subject may be a subject who has not been diagnosed by a doctor for coronavirus sequelae.
  • the subject of administration of the therapeutic agent according to one aspect of the present invention is not particularly limited, and may be humans or non-human mammals (eg, domestic animals, pets, and experimental animals).
  • Non-human mammals include, for example, monkeys, chimpanzees, cows, pigs, sheep, goats, horses, dogs, cats, rabbits, mice, and rats.
  • a therapeutic agent according to one aspect of the present invention can be administered to an administration subject by any administration route.
  • administration routes include oral administration, parenteral administration, transdermal administration, transmucosal administration, and intravenous administration. Therefore, the dosage form of the therapeutic agent according to one aspect of the present invention can be an internal medicine, an external medicine, an injection, or the like.
  • the route of administration of the therapeutic agent according to one aspect of the present invention is preferably oral administration, because administration is simple and the burden on the administration subject is small. Therefore, the dosage form of the therapeutic drug according to one aspect of the present invention is preferably an oral drug.
  • a therapeutic agent according to one aspect of the present invention can be formulated by a known method using an acetylcholine receptor agonist as an active ingredient and other ingredients as raw materials.
  • the therapeutic agent according to one aspect of the present invention may be administered such that the dose of the active ingredient, the acetylcholine receptor agonist, is 0.1 mg to 1000.0 mg/kg body weight. ⁇ 500.0 mg/kg body weight may be administered, 1.0 mg to 500.0 mg/kg body weight may be administered, and 1.0 mg to 300.0 mg/kg body weight may be administered.
  • 1.0 mg to 100.0 mg/kg body weight may be administered to 1.0 mg to 50.0 mg/kg body weight, 1.0 mg may be administered to be ⁇ 10.0 mg/kg, may be administered to be 1.0 to 10.0 mg/kg body weight, may be administered to be 1.0 to 5.0 mg/kg body weight may be administered.
  • the administration interval is, for example, once an hour, once every 1 to 6 hours, once every 6 to 12 hours, once every 12 hours to once a day, once every 1 to 3 days, once every 1 to 5 days. once a day, once every 1-7 days, once every 7-14 days, once every 14-21 days, once a month, once every 2 months, once every 3 months, It can be once every four months, once every five months, once every six months, or once a year.
  • the administration interval may be constant, but may be administered (abruptly) each time symptoms of the aftereffects of COVID-19 appear strongly.
  • the therapeutic agent according to one aspect of the present invention is administered, for example, once a day so that the dose of the active ingredient, the acetylcholine receptor agonist, is 1.0 to 5.0 mg/kg body weight. good too.
  • a screening method for a therapeutic drug for COVID-19 sequelae uses a COVID-19 sequelae model animal in which SARS-CoV-2 S1 protein is expressed in a non-human mammal, and treats COVID-19 sequelae.
  • a method of screening for a therapeutic drug for COVID-19 sequelae having a preventive effect wherein an administration step of administering a test substance to the COVID-19 sequelae model animal, and before and after administration of the test substance in the COVID-19 sequelae model animal, and an evaluation step of assessing changes in symptoms associated with the novel coronavirus. This makes it possible to screen drugs for the treatment of COVID-19 sequelae.
  • the method of screening for a drug for treating the sequelae of COVID-19 may be simply referred to as a "method of screening a drug.”
  • COVID-19 sequelae model animal used in the screening method for a therapeutic drug for COVID-19 sequelae according to one aspect of the present invention is an animal in which SARS-CoV-2 S1 protein is expressed in a non-human mammal.
  • the SARS-CoV-2 S1 protein may be transiently expressed or permanently expressed. Transgenic animals may also be used. Since it is possible to closely examine the symptoms at the time when the expression is terminated or attenuated, the expression of the SARS-CoV-2 S1 protein is transient, and at the time of use in the administration step of this screening method, in the COVID-19 sequelae model animal Expression of the SARS-CoV-2 S1 protein is preferably terminated or attenuated.
  • a model animal with the sequelae of COVID-19 is a model in which the SARS-CoV-2 S1 protein is expressed in at least one of the nasal and perinasal cavities of non-human mammals. Animals are preferred. In the COVID-19 sequelae model animal, inflammation may be induced prior to the step of administering the test substance.
  • the method for expressing the SARS-CoV-2 S1 protein in non-human mammals is not particularly limited.
  • a COVID-19 sequelae model animal can be produced by expressing the SARS-CoV-2 S1 protein in a non-human mammal according to the method for producing a COVID-19 sequelae model animal described below.
  • the administration step is a step of administering a test substance to a COVID-19 sequelae model animal.
  • the dose and administration method of the test substance are not particularly limited. It can be appropriately set according to the concentration of the active ingredient in the test substance, the dosage form of the test substance, and the like.
  • the evaluation step is a step of evaluating changes in symptoms associated with the novel coronavirus before and after administration of the test substance in the COVID-19 sequelae model animal.
  • the method of evaluating changes in symptoms related to the new coronavirus in COVID-19 sequelae model animals is not particularly limited.
  • the evaluation step it is possible to evaluate changes in symptoms associated with the novel coronavirus in the COVID-19 sequelae model animal by quantifying changes in the amount of behavior of the COVID-19 sequelae model animal.
  • inflammatory markers include inflammatory markers such as interleukin 6 (IL-6), tumor necrosis factor (TNF ⁇ ), chemokine CC motif ligand 2 (CCL2).
  • IL-6 interleukin 6
  • TNF ⁇ tumor necrosis factor
  • CCL2 chemokine CC motif ligand 2
  • the therapeutic or preventive effect of the test substance on the aftereffects of COVID-19 should be evaluated based on the evaluation results of changes in symptoms related to the new coronavirus.
  • the new coronavirus before and after administration of the test substance A test substance can be assessed as having activity in treating or preventing the sequelae of COVID-19 if the symptoms of virus-related fatigue or depression are ameliorated.
  • the evaluation process by quantifying the change in the expression level of inflammatory markers in the brain of the COVID-19 sequelae model animal, as a result of evaluating the change in symptoms related to the new coronavirus in the COVID-19 sequelae model animal, If the expression levels of inflammatory markers in the brain are significantly reduced before and after administration of the test substance, it can be evaluated that the test substance has an activity to treat or prevent the sequelae of COVID-19.
  • Screening by quantifying the change in the amount of behavior of the COVID-19 sequelae model animal and screening by quantifying the change in the expression level of inflammatory markers in the brain of the COVID-19 sequelae model animal can be used in combination as appropriate.
  • test substances can also be narrowed down.
  • Example 6 which will be described later, showed that acetylcholine is deficient in the brain affected by COVID-19. Therefore, acetylcholine receptor agonists can be high priority drug candidates.
  • Example 10 which will be described later, it was shown that among acetylcholine receptor agonists, PNU282987, an ⁇ 7 nicotinic receptor agonist that does not cross the blood-brain barrier, was administered to the brain ventricle and had the effect of suppressing brain inflammation.
  • ⁇ 7 nicotinic receptor agonists should be prioritized in future screening.
  • drugs that do not pass through the blood-brain barrier and therefore cannot be realistic candidates for therapeutic drugs can be used in methods such as intracerebroventricular administration, suggesting the feasibility of drug screening. showing.
  • Example 11 which will be described later, suggests that the mechanism is that the ⁇ 7 nicotinic receptor agonist increases the expression of the immunosuppressive molecule ZFP36. This result suggests that the clarification of the mechanism of the therapeutic effect of drugs in the screening process will enable the development of better efficacy assessment methods and the identification of drug target molecules.
  • a method for producing a COVID-19 sequelae model animal includes an expression step of expressing the SARS-CoV-2 S1 protein in a non-human mammal using a SARS-CoV-2 S1 protein expression vector. .
  • a COVID-19 sequelae model animal can be used as an experimental animal in the development of treatment or prevention methods for COVID-19 sequelae, particularly in the development of drugs such as therapeutic drugs and prophylactic drugs.
  • COVID-19 sequelae model animals can be used as experimental animals in research on the causes of the onset of COVID-19 sequelae. Therefore, it can contribute to Goal 3 of the Sustainable Development Goals (SDGs) "Good health and well-being for all”.
  • SDGs Sustainable Development Goals
  • the COVID-19 sequelae model animal produced by the method for producing a COVID-19 sequelae model animal according to one aspect of the present invention is a non-human mammal (mammal other than human) that can be used as an experimental animal.
  • a non-human mammal mammal other than human
  • the type of non-human mammal to be used in the expression step is not particularly limited, and can be appropriately selected depending on the intended use of the model animal to be produced.
  • Examples of non-human mammals used in the expression step include mice, rats, guinea pigs, dogs, rabbits, monkeys, and chimpanzees.
  • COVID-19 sequelae model animal is preferably a small animal model such as a mouse model.
  • the expression step is a step of expressing the SARS-CoV-2 S1 protein in a non-human mammal using a SARS-CoV-2 S1 protein expression vector.
  • the non-human mammals can develop COVID-19 sequelae.
  • Viral infection generally causes immune response to produce inflammatory cytokines, which is known to cause fatigue and depressive symptoms during the acute phase of infection.
  • the aftereffects of COVID-19 cause severe fatigue and depression that are not seen in other viruses, and that these symptoms persist as aftereffects. Therefore, the new coronavirus is expected to have a protein with strong activity that causes neuropathy upon infection. Therefore, the present inventors have made intensive studies to identify the protein, and as a result, the S1 region of the Spike protein (1273 amino acids, GenBank Accession No. YP 009724390) of the SARS-CoV-2 virus was found to be a sequelae of COVID-19. was found to be the causative protein of
  • the method for producing a COVID-19 sequelae model animal uses the SARS-CoV-2 S1 protein expression vector instead of the SARS-CoV-2 virus itself to produce the COVID-19 sequelae.
  • the SARS-CoV-2 S1 protein which is the causative protein of SARS-CoV-2, is expressed in non-human mammals. Therefore, a model animal produced by the method for producing a model animal with sequelae of COVID-19 according to one aspect of the present invention can be used in a normal experimental environment, and is easy to handle.
  • the animal model produced by the method for producing a model animal with sequelae of COVID-19 according to one aspect of the present invention is excellent in that the model animal with sequelae of COVID-19 can be obtained efficiently and reliably.
  • using the SARS-CoV-2 S1 protein expression vector to express the SARS-CoV-2 S1 protein in a non-human mammal means using the SARS-CoV-2 S1 protein expression vector to It means expressing the SARS-CoV-2 S1 protein in the non-human mammal of interest.
  • the site where the SARS-CoV-2 S1 protein is expressed in the body of the target non-human mammal is not particularly limited. It is preferred to express the SARS-CoV-2 S1 protein in at least one of the nasal cavity and the perinasal cavity. In order to express the SARS-CoV-2 S1 protein in at least one of the nasal and perinasal cavities of the non-human mammal, for example, in the expression step, the SARS-CoV-2 S1 protein expression vector is introduced into the nasal cavity of the non-human mammal. may be administered.
  • the structure of the SARS-CoV-2 Spike protein is shown in Figure 1.
  • the S1 region is a region having an amino acid sequence consisting of the 1st to 685th amino acids in the amino acid sequence of the SARS-CoV-2 Spike protein.
  • the S1 region contains a signal peptide sequence (SP), an N-terminal domain (NTD) and a receptor binding domain (RBD).
  • SP signal peptide sequence
  • NTD N-terminal domain
  • RBD receptor binding domain
  • a polypeptide comprising the S1 region of the Spike protein of the SARS-CoV-2 virus is referred to herein as "SARS-CoV-2 S1 protein" or simply "S1 protein.”
  • the SARS-CoV-2 S1 protein may be, for example, a polypeptide of (a) or (b): (a) A polypeptide having an amino acid sequence (SEQ ID NO: 1) consisting of the 1st to 685th amino acids in the amino acid sequence shown in GeneBank Accession No. YP_009724390; (B) of the amino acid sequence shown in GeneBank Accession No. YP_009724390, consisting of an amino acid sequence (SEQ ID NO: 1) consisting of the 1st to 685th amino acids and an amino acid sequence with a sequence identity of 80% or more, and in cells A polypeptide having the activity of increasing intracellular calcium concentration upon introduction.
  • the SARS-CoV-2 S1 protein is a polypeptide with a molecular weight of about 76.7 kDa, consisting of 685 amino acids.
  • the SARS-CoV-2 S1 protein like the polypeptide (b) above, has an amino acid sequence (SEQ ID NO: 1 ) and an amino acid sequence with a sequence identity of 80% or more (85% or more, 90% or more, 95% or more, 98% or more, 99% or more), and have the activity of increasing intracellular calcium concentration when introduced into cells. It may be a polypeptide having As used herein, percent identity of amino acid sequences is calculated using genetic information processing software GENETYX Ver. 7 (manufactured by Genetics).
  • polypeptide of (b) has 100 or less amino acids in the amino acid sequence (SEQ ID NO: 1) consisting of the 1st to 685th amino acids in the amino acid sequence shown in GeneBank Accession No. YP_009724390. It may be a polypeptide consisting of a substituted, deleted, inserted and/or added amino acid sequence and having the activity of increasing intracellular calcium concentration when introduced into a cell.
  • substitution, deletion, insertion, and/or addition of 100 or less amino acids means 100 or less (90 80 or less, 70 or less, 60 or less, 50 or less, 40 or less, 30 or less, 20 or less, 15 or less, 10 or less, 7 or less, 5 or less, or 2 or less ) are substituted, deleted, inserted and/or added.
  • the polypeptide of (b) can be said to be a variant of the polypeptide of (a).
  • the term "mutation" as used herein mainly means a mutation artificially introduced by a known method for producing a mutant protein, but it may be one obtained by isolating and purifying a naturally occurring similar mutant protein.
  • SARS-CoV-2 B 1.1.7 strains (so-called “alpha strains”): deletion69-70, deletion144-145, N501Y, A570D, and D614G, P681H - SARS-CoV-2 B.
  • 1.351 strains (so-called “beta strains”): D80A, D215G, Deletion241-243, K417N, E484K, N501Y, and D614G ⁇ SARS-CoV-2P.
  • One lineage (so-called "gamma strain”): L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N501Y, D614G, and H655Y - SARS-CoV-2 B.
  • strains T19R, G142D, E156G, deletion157-158, L452R, T487K, E484Q, D614G, and P681R - SARS-CoV-2 B.
  • 1.1.529 strains (so-called "Omicron strains"): G142D, G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, D614G, H 655Y , N679K, and P681H
  • the SARS-CoV-2 S1 protein mutant protein has the activity of increasing intracellular calcium concentration, which was confirmed by expressing the mutant protein in arbitrary cultured cells and measuring the intracellular calcium concentration. can do.
  • the intracellular calcium concentration can be measured by the method described in Examples below. When the calcium concentration in the mutant protein-expressing cells is significantly increased compared to the parent cell, it can be determined that the mutant protein has the activity of increasing the intracellular calcium concentration.
  • a SARS-CoV-2 S1 protein expression vector is an expression vector obtained by introducing a polynucleotide encoding the SARS-CoV-2 S1 protein into a known plasmid vector or viral vector.
  • plasmid vector or viral vector As the plasmid vector or viral vector, as long as the SARS-CoV-2 S1 protein can be expressed in the body of a non-human mammal, a known vector commonly used in genetic recombination can be appropriately selected. Not limited. Viral vectors can be preferably used because of their high infection efficiency and the ease of introduction into non-human mammals. As an example of a virus vector, an adenovirus vector that is commonly used for purposes such as known gene therapy and virus vaccines can be particularly preferably used.
  • the type of the promoter for expressing the SARS-CoV-2 S1 protein is not particularly limited as long as it can express mRNA in cells infected with SARS-CoV-2.
  • a promoter that allows SARS-CoV-2 S1 protein to be produced to the same extent as during SARS-CoV-2 infection can be particularly preferably used.
  • a polynucleotide encoding the SARS-CoV-2 S1 protein may be, for example, a polynucleotide encoding the polypeptide (a) or (b) described above.
  • the entire genome sequence of SARS-CoV-2 S1 including the base sequence of the polynucleotide encoding the polypeptide of (a), is published in GenBank Accession No. MN908947.
  • the polynucleotide encoding the polypeptide of (a) has a base sequence (SEQ ID NO: 2) consisting of nucleotides 21563-23617 in the base sequence shown in GeneBank Accession No. MN908947.
  • the polynucleotide encoding the polypeptide of (a) has a size of 2055 base pairs (about 2 kbp).
  • the method for obtaining the polynucleotide encoding the SARS-CoV-2 S1 protein is not particularly limited.
  • a method using amplification means such as PCR can be mentioned.
  • primers are prepared from the 5' and 3' sequences (or their complementary sequences), and these primers are used to (or cDNA) as a template to amplify the DNA region sandwiched between the two primers, a large amount of DNA fragments containing the polynucleotide encoding the SARS-CoV-2 S1 protein can be obtained.
  • a polynucleotide having the base sequence of the polynucleotide encoding the SARS-CoV-2 S1 protein may be synthesized using known chemical synthesis.
  • a base sequence optimized for the codon usage of the animal to be used may be used.
  • the SARS-CoV-2 S1 protein may be transiently expressed in a non-human mammal, or may be permanently expressed. good too.
  • the method for producing a COVID-19 sequelae model animal according to one aspect of the present invention preferably further includes an inflammation-inducing step of inducing inflammation in the non-human mammal.
  • the SARS-CoV-2 S1 protein is expressed in inflammatory conditions due to SARS-CoV-2 virus infection. Therefore, the method for producing a COVID-19 sequelae model animal according to one aspect of the present invention further includes an inflammation-inducing step, so that it is possible to produce a model animal that takes into consideration the situation of actual SARS-CoV-2 virus infection. can.
  • the method of inducing inflammation in non-human mammals is not particularly limited, but from the viewpoint of the ease of handling of the model animals produced, it is preferable to induce inflammation not by viral infection but by drugs or the like.
  • inflammation can be induced in non-human mammals by intraperitoneal administration of lipopolysaccharides (LPS) derived from Gram-negative bacteria such as E. coli O111.
  • LPS is known to have the biological activity of activating macrophages.
  • the inflammation induction process is performed after the expression process.
  • the inflammation-inducing step By performing the inflammation-inducing step after the expression step, the inflammation-inducing effect can be observed in model animals expressing COVID-19 sequelae.
  • the inflammation-inducing step may precede the expressing step.
  • the method for producing a COVID-19 sequelae model animal may further include an evaluation step of evaluating the degree of symptoms of COVID-19 sequelae in the non-human mammal after the expression step.
  • COVID-19 sequelae The typical symptoms of COVID-19 sequelae are fatigue and depression. Therefore, behavioral experiments and their quantification can assess the severity of symptoms of COVID-19 sequelae in non-human mammals after the onset process by assessing the severity of fatigue or depressive symptoms.
  • the degree of fatigue can be evaluated by conducting a forced swimming test with a weight of 10% shown in the examples below. Further, the degree of depressive symptoms can be evaluated by conducting a tail suspension test shown in Examples described later.
  • the model animal produced by the method for producing a model animal with sequelae of COVID-19 according to one aspect of the present invention does indeed exhibit symptoms of sequelae of COVID-19.
  • the amount of SARS-CoV-2 S1 protein to be expressed in the expression step, the type of expression vector, the degree of inflammation to be induced in the inflammation induction step, and the like can be adjusted as appropriate.
  • a COVID-19 sequelae animal model production kit (hereinafter simply “kit”) according to one aspect of the present invention includes an expression vector capable of expressing SARS-CoV-2 S1 protein in cells of non-human mammals.
  • a kit according to one aspect of the present invention can be suitably used in a method for producing a model animal for the sequelae of COVID-19 according to one aspect of the present invention.
  • kits are intended to be a package that includes a container (eg, bottle, plate, tube, dish, etc.) containing specific materials.
  • the kit according to one aspect of the present invention may be in a form in which each material contained therein exists independently, or in a form in which a plurality of materials are mixed (e.g., in the form of a composition) There may be.
  • Kits preferably include instructions for using each material.
  • the kit according to one aspect of the present invention only needs to include materials for carrying out the method for producing a COVID-19 sequelae model animal according to one aspect of the present invention, and SARS-CoV in the cells of non-human mammals.
  • materials for carrying out the method for producing a COVID-19 sequelae model animal according to one aspect of the present invention and SARS-CoV in the cells of non-human mammals.
  • SARS-CoV in the cells of non-human mammals.
  • the specific construction of the kit, materials, equipment, etc. are not particularly limited.
  • the kit according to one aspect of the present invention contains an expression vector capable of expressing the SARS-CoV-2 S1 protein in non-human mammalian cells, and lipopolysaccharide (LPS) for use in the inflammation-inducing step. You may have more.
  • LPS lipopolysaccharide
  • COVID-19 sequelae model animals A COVID-19 sequelae model animal produced by the method for producing a COVID-19 sequelae model animal according to one aspect of the present invention is also included in the scope of the present invention. Since the COVID-19 sequelae model animal according to one aspect of the present invention is produced by the method for producing a COVID-19 sequelae model animal according to one aspect of the present invention, it does not require a high-level containment facility such as a P3 facility, Since it can be used in a normal experimental environment, it is easy to handle.
  • a high-level containment facility such as a P3 facility
  • one aspect of the present invention is as follows.
  • a therapeutic drug for aftereffects of novel coronavirus infection containing an acetylcholine receptor agonist as an active ingredient.
  • the acetylcholine receptor agonist is donepezil.
  • ⁇ 4> The novel coronavirus infection sequelae therapeutic drug according to any one of ⁇ 1> to ⁇ 3>, wherein the novel coronavirus infection sequelae is fatigue associated with the novel coronavirus infection.
  • ⁇ 5> The therapeutic drug for aftereffects of novel coronavirus infection according to any one of ⁇ 1> to ⁇ 3>, wherein the aftereffects of novel coronavirus infection are depressive symptoms associated with the novel coronavirus.
  • ⁇ 6> The therapeutic drug for aftereffects of novel coronavirus infection according to any one of ⁇ 1> to ⁇ 3>, wherein the aftereffects of novel coronavirus infection are olfactory disorders associated with the novel coronavirus.
  • ⁇ 7> The therapeutic drug for aftereffects of novel coronavirus infection according to any one of ⁇ 1> to ⁇ 3>, wherein the aftereffects of novel coronavirus infection are memory impairment associated with the novel coronavirus.
  • the therapeutic drug screening method according to ⁇ 8>, wherein the model animal is a model animal that expresses the SARS-CoV-2 S1 protein in at least one of the nasal and perinasal cavities of the non-human mammal. . ⁇ 10>
  • the model animal includes an expression step of expressing the SARS-CoV-2 S1 protein in the non-human mammal using a SARS-CoV-2 S1 protein expression vector.
  • the therapeutic agent screening method according to ⁇ 8> or ⁇ 9> which is a model animal produced by the production method of ⁇ 8> or ⁇ 9>.
  • a method for producing a novel coronavirus infection sequelae model animal comprising an expression step of expressing the SARS-CoV-2 S1 protein in a non-human mammal using a SARS-CoV-2 S1 protein expression vector.
  • YP_009724390 (B) of the amino acid sequence shown in GeneBank Accession No. YP_009724390, consisting of an amino acid sequence (SEQ ID NO: 1) consisting of the 1st to 685th amino acids and an amino acid sequence with a sequence identity of 80% or more, and in cells A polypeptide having the activity of increasing intracellular calcium concentration upon introduction.
  • Method for treating or preventing sequelae of COVID-19> Also included within the scope of the present invention is a method of treating or preventing COVID-19 sequelae using the COVID-19 sequelae therapeutic agent according to one aspect of the present invention.
  • a method for treating or preventing the sequelae of COVID-19 is as follows.
  • a method for treating or preventing the aftereffects of COVID-19 which comprises administering to a subject (e.g., human or non-human animal) a drug for treating the aftereffects of COVID-19, which contains an acetylcholine receptor agonist as an active ingredient.
  • a subject e.g., human or non-human animal
  • acetylcholine receptor agonist is a central acetylcholine receptor agonist that acts on acetylcholine receptors in the brain.
  • ⁇ 18> The method for treating or preventing sequelae of COVID-19 according to ⁇ 16> or ⁇ 17>, wherein the acetylcholine receptor agonist is donepezil.
  • ⁇ 19> The method for treating or preventing the aftereffects of COVID-19 according to any one of ⁇ 16> to ⁇ 18>, wherein the aftereffects of COVID-19 are fatigue associated with the novel coronavirus.
  • ⁇ 20> The method for treating or preventing the aftereffects of COVID-19 according to any one of ⁇ 16> to ⁇ 18>, wherein the aftereffects of COVID-19 are depressive symptoms associated with the novel coronavirus.
  • ⁇ 21> The method for treating or preventing the aftereffects of COVID-19 according to any one of ⁇ 16> to ⁇ 18>, wherein the aftereffects of COVID-19 are olfactory disorders associated with the novel coronavirus.
  • ⁇ 22> The method for treating or preventing the aftereffects of COVID-19 according to any one of ⁇ 16> to ⁇ 18>, wherein the aftereffects of COVID-19 are memory disorders associated with the novel coronavirus.
  • the administration target, administration route, formulation, prescription, etc. of the therapeutic drug for the aftereffects of COVID-19 are as described for the therapeutic drug according to one aspect of the present invention, and will not be repeated here.
  • Oral administration of therapeutic agents for the aftereffects of COVID-19 is preferred because administration is simple and less of a burden on the administration subject.
  • uses according to one aspect of the present invention are as follows.
  • ⁇ 23> Use of an acetylcholine receptor agonist for the manufacture of a therapeutic drug for the sequelae of COVID-19.
  • ⁇ 24> The use according to ⁇ 23>, wherein the acetylcholine receptor agonist is a central acetylcholine receptor agonist that acts on acetylcholine receptors in the brain.
  • ⁇ 25> The use according to ⁇ 23> or ⁇ 24>, wherein the acetylcholine receptor agonist is donepezil.
  • ⁇ 26> The use according to any one of ⁇ 23> to ⁇ 25>, wherein the COVID-19 sequela is fatigue associated with the novel coronavirus.
  • ⁇ 27> The use according to any one of ⁇ 23> to ⁇ 25>, wherein the aftereffects of COVID-19 are depressive symptoms associated with the novel coronavirus.
  • ⁇ 28> The use according to any one of ⁇ 23> to ⁇ 25>, wherein the aftereffect of COVID-19 is an olfactory disorder associated with the novel coronavirus.
  • ⁇ 29> The use according to any one of ⁇ 23> to ⁇ 25>, wherein the aftereffect of COVID-19 is memory impairment associated with novel coronavirus.
  • Example 1 The neurotoxicity of the SARS-CoV-2 S1 protein was examined using intracellular calcium elevation as an index.
  • S1 protein is an amino acid sequence consisting of the 1st to 685th amino acids (SEQ ID NO: 1).
  • SEQ ID NO: 1 The nucleotide sequence of the DNA fragment encoding the S1 protein is published under GenBank Accession No. MN908947.
  • Adenovirus Dual Expression Kit (TaKaRa) was used to construct the S1-expressing adenovirus vector.
  • a DNA fragment encoding the S1 region was incorporated into the cosmid vector pAxCAwtit2 to construct an adenovirus vector (S1/Adv) expressing S1 under the control of the CAG promoter.
  • Mouse skin-derived fibroblast cell line 3T3 cell line and human alveolar basal epithelial adenocarcinoma cell line A549 were used to express S1 protein in mouse and human cells.
  • CalPhos Mammalian Transfection Kit (TaKaRa) was used to introduce S1/pFlag into these cells to transiently express S1 protein.
  • Empty vector plasmid pFlag-CMV-5a was used as a control.
  • adenovirus vector In the case of the adenovirus vector, these cells were directly infected with S1/Adv to transiently express the S1 protein. An empty adenoviral vector was used as a control.
  • the S1 protein used for constructing the expression vector is the amino acid sequence of the SARS-CoV-2 Spike protein shown in GeneBank Accession No. YP_009724390.
  • a protein having the sequence SEQ ID NO: 1).
  • 3T3 cells and A549 cells were transformed with the S1 protein expression plasmid or control plasmid, and the intracellular calcium concentration was measured.
  • the results are shown in FIG.
  • "Intensity/area” shown on the vertical axis of the graph in FIG. 2 represents fluorescence intensity per unit area.
  • Control shown on the horizontal axis of the graph in FIG. 2 represents cells transformed with the control plasmid
  • S1/pFlag represents cells transformed with the S1 protein expression plasmid.
  • the horizontal bar shown in FIG. 2 represents the median value. ***: P ⁇ 0.0001.
  • the intracellular calcium concentration increased due to the expression of the S1 protein in all cells.
  • ⁇ Fatigue behavior test> Six days after nasal administration of S1/Adv or vector/Adv, a forced swimming test with a weight of 10% was performed as a fatigue behavior test. As a method, the body weights of S1 mice and control mice were measured in the morning of the day of the test, and a weight of about 10% of the body weight was prepared. The weight was fixed to the tail of S1 mice or control mice, placed in a water tank for forced swimming test, and the time until the nose tip was submerged under water for 10 seconds was measured.
  • S1 mice Compared to control mice, S1 mice tended to have shorter swimming times. This indicates that S1 mice tire more easily than control mice.
  • Example 3 Expression of depression-like behavior in S1 mice ⁇ Method> S1 mice and control mice were produced by the same method as in Example 2. A tail suspension test was performed to confirm depression-like behavior. As a method, the tails of S1 mice and control mice on the 6th day after nasal administration of S1 / Adv or vector / Adv were fixed, suspended for 10 minutes, recorded, and analyzed with image analysis software TailSuspScan (CleverSys Inc). and the motionless time was measured.
  • FIG. 5 shows the results of plotting the immobility time of the tail suspension test.
  • Control shown on the horizontal axis of the graph in FIG. 5 represents control mice, and "S1" represents S1 mice. *: P ⁇ 0.05.
  • Example 4 Olfactory bulb nerve damage in mice expressing S1 protein and induced inflammation by LPS ⁇ Method> S1 mice and control mice were produced by the same method as in Example 2. Escherichia coli O111-derived lipopolysaccharide (MERCK) was intraperitoneally administered to S1 mice and control mice on day 7 after intranasal administration of S1/Adv or vector/Adv at 5 mg/kg, and olfactory bulbs were detected 30 and 60 minutes after intraperitoneal administration. was taken.
  • MERCK Escherichia coli O111-derived lipopolysaccharide
  • RNA was purified with the RNeasy Mini Kit (QIAGEN). Using the purified RNA as a template, cDNA was synthesized using PrimeScript RT reagent Kit (Takara Bio). Gene expression of calbindin was analyzed by RT-qPCR using the synthesized cDNA. The measurement results of 18S rRNA were used for normalization.
  • the amount of calbindin expression in the olfactory bulb of S1 mice tended to decrease 30 minutes after LPS administration, and significantly decreased 60 minutes after LPS administration. Since calbindin is a marker of mature neurons, it was thought that mature neurons in the olfactory bulb of S1 mice were damaged when peripheral inflammation was induced.
  • Example 5 Cranial nerve damage in mice in which S1 protein was expressed and inflammation was induced by LPS ⁇ Method> S1 mice and control mice were produced by the same method as in Example 2.
  • E. coli O111-derived lipopolysaccharide (MERCK) was intraperitoneally administered to S1 mice and control mice on day 7 after intranasal administration of S1/Adv or vector/Adv, and 5 mg/kg of lipopolysaccharide (MERCK) was administered intraperitoneally. Taken.
  • RNA was purified with the RNeasy Mini Kit (QIAGEN). Using the purified RNA as a template, cDNA was synthesized using PrimeScript RT reagent Kit (Takara Bio). Gene expression of calbindin was analyzed by RT-qPCR using the synthesized cDNA. The measurement results of 18S rRNA were used for normalization.
  • Brains were excised from S1 mice and control mice on day 7 after intranasal administration of S1/Adv or vector/Adv and fixed with a 10% neutral formalin solution.
  • the fixed brain was embedded in paraffin, and a coronal section (brain section) was prepared at a position (Bregma 0.62 mm) where the septal area and Broca's diagonal band can be observed.
  • the prepared brain sections were subjected to antigen retrieval treatment after deparaffinization, and fluorescent immunostaining was performed with an Anti-Choline Acetyltransferase antibody (manufactured by Abcam).
  • FIG. 8 shows the results of fluorescent immunostaining. From FIG. 8, in S1 mice, cholinergic neurons, which are choline acetyltransferase (ChAT)-positive cells, in the basal forebrain septal area (MS) and Broca's diagonal zone (DB) compared to control mice. suggested that the numbers were declining. Therefore, the number of ChAT-positive cells in the MS/DB region was counted. The results are shown in FIG. FIG. 9 showed that the number of ChAT-positive cells in the MS/DB region was significantly reduced in S1 mice. **: P ⁇ 0.01.
  • ChAT choline acetyltransferase
  • Example 7 Improvement of fatigue symptoms in S1 mice by administration of donepezil ⁇ Method> ⁇ Administration of donepezil> Donepezil (Fujifilm Wako Pure Chemical Industries, Ltd.) was dissolved in water to a concentration of 32 mg/L, and administered to mice through drinking water. This brings the donepezil dose to mice to 4.0 mg/kg/day. Since there are many literatures on donepezil doses of 3.0 to 5.0 mg/kg/day, 4.0 mg/kg/day was adopted.
  • FIG 10 shows the administration scheme of donepezil.
  • the donepezil solution was administered to S1 and control mice immediately after intranasal administration of S1/Adv or vector/Adv, and the donepezil-containing drinking water was changed once every two days.
  • Donepezil was also administered in the following examples in the same manner.
  • ⁇ Fatigue behavior test> Six days after nasal administration of S1/Adv or vector/Adv, a forced swimming test with a weight of 10% was performed as a fatigue behavior test. As a method, the body weights of S1 mice and control mice were measured in the morning of the day of the test, and a weight of about 10% of the body weight was prepared. The weights were fixed to the tails of S1 mice and control mice, placed in a water tank for forced swimming test, and the time it took for the tip of the nose to sink under the water surface for 10 seconds was measured.
  • Example 8 Improvement of depression-like behavior in S1 mice by administration of donepezil ⁇ Method> After intranasal administration of S1/Adv or vector/Adv, a tail suspension test was performed to confirm depression-like behavior in S1 mice and control mice receiving donepezil in drinking water according to the administration scheme shown in FIG. .
  • the tails of S1 mice and control mice on the 6th day after nasal administration of S1 / Adv or vector / Adv were fixed, suspended for 10 minutes, recorded, and analyzed with image analysis software TailSuspScan (CleverSys Inc). and the motionless time was measured.
  • FIG. 12 shows the results of plotting the immobility time of the tail suspension test.
  • Control shown on the horizontal axis of the graph in FIG. 12 represents control mice, and "S1" represents S1 mice. *: P ⁇ 0.05, ***: P ⁇ 0.001.
  • Example 9 Improvement of brain inflammation in S1 mice by administration of donepezil ⁇ Method> After intranasal administration of S1/Adv or vector/Adv, in S1 mice and control mice that were administered donepezil in drinking water according to the administration scheme shown in FIG. Other brains were collected. Using the harvested brain, RNA was purified with RNeasy Mini Kit (QIAGEN). Using the purified RNA as a template, cDNA was synthesized using PrimeScript RT reagent Kit (Takara Bio).
  • IL-6 interleukin 6
  • TNF ⁇ tumor necrosis factor
  • CCL2 chemokine CC motif ligand 2
  • IL-6 expression in the brain of S1 mice was significantly increased in the group to which donepezil was not administered (“-” in FIG. 13), and other TNF ⁇ and CCL2 levels were significantly increased. An increasing trend was also observed in the expression.
  • the expression of these genes in the brain of S1 mice tended to decrease, and the expression of these genes in the brain of control mice showed a difference. was not accepted.
  • brain inflammation was induced in S1 mice due to S1 protein expression, and administration of donepezil ameliorated brain inflammation.
  • Example 10 Evaluation of the effect of acetylcholine receptor agonists other than donepezil ⁇ Method> Since donepezil exerts its effects by increasing the amount of acetylcholine in the brain, in order to develop more effective therapeutic agents, agents that act more selectively on acetylcholine receptors were used. Screening is required. Therefore, in S1 mice and control mice, PNU282987, an ⁇ 7 nicotinic acetylcholine receptor-specific agonist, was intracerebroventricularly administered at 400 nmol/mouse on day 7 after intranasal administration of S1/Adv or vector/Adv. , examined the therapeutic effect on brain inflammation in S1 mice.
  • IL-1 ⁇ interleukin 1 ⁇
  • IL-6 interleukin 6
  • Example 11 Examples of Action Mechanisms of Acetylcholine Receptor Agonists ⁇ Method>
  • Zfp36 zinc finger protein 36
  • a drug for treating the aftereffects of COVID-19 according to one aspect of the present invention can contribute to the treatment or prevention of the aftereffects of COVID-19 in patients infected with the novel coronavirus.
  • the screening method for a drug for treating the aftereffects of COVID-19 according to one aspect of the present invention can contribute to the development of new drugs for treating the aftereffects of COVID-19.
  • a COVID-19 sequelae model animal produced by the method for producing a COVID-19 sequelae model animal according to one aspect of the present invention can be used for drug development and basic research on COVID-19 sequelae.

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Abstract

Provided is a drug for treating aftereffects of a novel coronavirus infection. The drug for treating aftereffects of the novel coronavirus infection contains an acetylcholine receptor agonist as an active ingredient.

Description

新型コロナウイルス感染症後遺症の治療薬Drugs for the aftereffects of COVID-19
　本発明は、新型コロナウイルス感染症後遺症の治療薬、新型コロナウイルス感染症後遺症の治療薬のスクリーニング方法、及び新型コロナウイルス感染症後遺症モデル動物の製造方法に関する。 The present invention relates to a therapeutic drug for the aftereffects of a new coronavirus infection, a method for screening a therapeutic drug for the aftereffects of a new coronavirus infection, and a method for producing a model animal for the aftereffects of a new coronavirus infection.
　ＳＡＲＳ－ＣｏＶ－２ウイルスによって引き起こされる急性呼吸器疾患ＣＯＶＩＤ－１９（いわゆる、新型コロナウイルス感染症）は、世界中で多くの感染者を発生させている重篤な感染症である。 The acute respiratory disease COVID-19 (so-called novel coronavirus infection) caused by the SARS-CoV-2 virus is a serious infectious disease that has caused many infected people around the world.
　ＣＯＶＩＤ－１９は急性期に重篤な疾患を発生させることで患者を死に至らしめる他、回復期に高頻度で後遺症（以下、「新型コロナウイルス感染症後遺症」）を生じることが知られている。新型コロナウイルス感染症後遺症のなかで特に頻度が高いのは、疲労、うつ症状、嗅覚障害であると言われている。また、新型コロナウイルス感染症後遺症は急性期の症状があまり重篤でない患者にも発生するために、ワクチンによって重症化が抑制された場合でも発生する可能性が高いと考えられる。 COVID-19 is known to cause death in patients by causing serious illness in the acute phase, and to frequently cause aftereffects (hereinafter referred to as "new coronavirus infection sequelae") during the recovery period. . Among the aftereffects of COVID-19, fatigue, depression, and olfactory disturbance are said to be particularly common. In addition, since the aftereffects of COVID-19 can occur even in patients with less severe symptoms in the acute phase, it is highly likely that they will occur even if the severity of the symptoms is suppressed by the vaccine.
　新型コロナウイルス感染症後遺症は、その名が示すとおりウイルス感染の後遺症であるので、後遺症の発生時には原因ウイルスであるＳＡＲＳ－ＣｏＶ－２ウイルスの増殖は生じていない。このため、ウイルス増殖を抑制可能な薬剤を新型コロナウイルス感染症後遺症の発生後に投与しても後遺症に対する効果は期待できない。このため、新型コロナウイルス感染症後遺症に対する治療薬は、ＳＡＲＳ－ＣｏＶ－２ウイルスの感染によって生じた組織の障害を修復するものである必要があると考えられる。 As the name suggests, the aftereffects of the new coronavirus infection are the aftereffects of viral infection, so when the aftereffects occur, the SARS-CoV-2 virus, the causative virus, does not proliferate. Therefore, even if a drug capable of suppressing virus proliferation is administered after the onset of the aftereffects of the new coronavirus infection, it cannot be expected to be effective against the aftereffects. Therefore, it is thought that therapeutic agents for the aftereffects of novel coronavirus infection need to repair tissue damage caused by SARS-CoV-2 virus infection.
　新型コロナウイルス感染症後遺症の治療に関しては、現在有効なものがない。このため、根拠のない民間療法が横行しており、健康被害も出ている。 There is currently no effective treatment for the aftereffects of the new coronavirus infection. For this reason, unfounded folk remedies are rampant, and there are health hazards.
　新型コロナウイルス感染症後遺症の治療薬が求められている。しかし、有効な治療薬はない。治療薬開発のために新型コロナウイルス感染症後遺症の研究を行うためには、新型コロナウイルス感染症後遺症の症状を呈する動物モデルが重要である。しかし、これまでの新型コロナウイルスに関する動物モデルは、感染予防と急性症状に関する研究が中心であったため、後遺症研究に適する動物モデル開発は十分ではない。 There is a demand for a therapeutic drug for the aftereffects of the new coronavirus infection. However, there are no effective therapeutic agents. Animal models exhibiting the symptoms of the aftereffects of the new coronavirus infection are important for research on the aftereffects of the new coronavirus infection for the development of therapeutic drugs. However, animal models related to the new coronavirus have focused on research on infection prevention and acute symptoms, so the development of animal models suitable for sequelae research is not sufficient.
　本発明の一態様は、新型コロナウイルス感染症後遺症治療薬を提供することを課題とする。 An object of one aspect of the present invention is to provide a therapeutic drug for the aftereffects of the novel coronavirus infection.
　本発明者らは、上記の課題を解決するために鋭意検討した結果、ＳＡＲＳ－ＣｏＶ－２ウイルスのＳｐｉｋｅタンパク質のＳ１領域が、新型コロナウイルス感染症後遺症の原因タンパク質であることを初めて見出し、新型コロナウイルス感染症後遺症の症状を呈する動物モデルを完成させるに至った。本発明者らは、新型コロナウイルス感染症後遺症モデル動物を用いた新型コロナウイルス感染症後遺症の研究の結果、新型コロナウイルス感染症後遺症において脳内のアセチルコリンが減少していることと、受容体作動薬によって新型コロナウイルス感染症後遺症を治療又は予防できることと、を初めて見出した。 As a result of intensive studies to solve the above problems, the present inventors found for the first time that the S1 region of the Spike protein of the SARS-CoV-2 virus is the causative protein of the aftereffects of the new coronavirus infection. We have completed an animal model that exhibits symptoms of the aftereffects of coronavirus infection. As a result of research on the aftereffects of the new coronavirus infection using animal models of the aftereffects of the new coronavirus infection, the present inventors found that acetylcholine in the brain is reduced in the aftereffects of the new coronavirus infection, and receptor activation For the first time, we discovered that drugs can treat or prevent the aftereffects of COVID-19.
　すなわち、本発明の一態様は、アセチルコリン受容体作動薬を有効成分として含む、新型コロナウイルス感染症後遺症治療薬、である。 That is, one aspect of the present invention is a drug for treating sequelae of novel coronavirus infection, which contains an acetylcholine receptor agonist as an active ingredient.
　また、本発明の一態様は、非ヒト哺乳動物にＳＡＲＳ－ＣｏＶ－２　Ｓ１タンパク質を発現させた新型コロナウイルス感染症後遺症モデル動物に、試験物質を投与する投与工程と、
　前記モデル動物における、前記試験物質の投与前後の、新型コロナウイルスが関与する症状の変化を評価する評価工程と、を含む新型コロナウイルス感染症後遺症治療薬のスクリーニング方法、である。 In addition, one aspect of the present invention is an administration step of administering a test substance to a novel coronavirus infection sequelae model animal in which SARS-CoV-2 S1 protein is expressed in a non-human mammal;
and an evaluation step of evaluating changes in symptoms related to the novel coronavirus before and after administration of the test substance in the model animal.
　また、本発明の一態様は、ＳＡＲＳ－ＣｏＶ－２　Ｓ１タンパク質発現ベクターを用いて当該ＳＡＲＳ－ＣｏＶ－２　Ｓ１タンパク質を非ヒト哺乳動物に発現させる発現工程を含む、新型コロナウイルス感染症後遺症モデル動物の製造方法、である。 In addition, one aspect of the present invention is a novel coronavirus infection sequelae model animal, comprising an expression step of expressing the SARS-CoV-2 S1 protein in a non-human mammal using a SARS-CoV-2 S1 protein expression vector. manufacturing method.
　本発明の一態様によれば、新型コロナウイルス感染症後遺症の治療薬を提供することができる。 According to one aspect of the present invention, it is possible to provide a therapeutic drug for the aftereffects of novel coronavirus infection.
ＳＡＲＳ－ＣｏＶ－２　Ｓｐｉｋｅタンパク質の構造を示す模式図である。Schematic diagram showing the structure of the SARS-CoV-2 Spike protein. 実施例の結果を示し、Ｓ１タンパク質発現プラスミド又は対照プラスミドを形質転換した３Ｔ３細胞及びＡ５４９細胞における細胞内カルシウム濃度を測定した結果を示す図である。FIG. 3 shows the results of Examples, and shows the results of measuring intracellular calcium concentrations in 3T3 cells and A549 cells transformed with an S1 protein expression plasmid or a control plasmid. 実施例の結果を示し、Ｓ１タンパク質発現アデノウイルス又は対照アデノウイルスを感染させた３Ｔ３細胞及びＡ５４９細胞における細胞内カルシウム濃度を測定した結果を示す図である。FIG. 3 shows the results of Examples and shows the results of measuring intracellular calcium concentration in 3T3 cells and A549 cells infected with S1 protein-expressing adenovirus or control adenovirus. 実施例の結果を示し、対照マウス及びＳ１タンパク質発現マウス（Ｓ１マウス）における１０％の重り付き強制水泳試験の結果を示す図である。FIG. 10 shows the results of the Examples and shows the results of the 10% weighted forced swimming test in control mice and S1 protein-expressing mice (S1 mice). 実施例の結果を示し、対照マウス及びＳ１マウスにおける尾懸垂試験の結果を示す図である。FIG. 4 shows the results of the Examples and shows the results of the tail suspension test in control and S1 mice. 実施例の結果を示し、対照マウス及びＳ１マウスにおけるＬＰＳ投与後の嗅球におけるカルビンジン遺伝子発現量を解析した結果を示す図である。FIG. 10 shows the results of Examples, and shows the results of analyzing the calbindin gene expression level in the olfactory bulb after LPS administration in control mice and S1 mice. 実施例の結果を示し、対照マウス及びＳ１マウスにおけるＬＰＳ投与後の脳におけるカルビンジン遺伝子発現量を解析した結果を示す図である。FIG. 10 shows the results of Examples, and shows the results of analyzing the calbindin gene expression level in the brain after administration of LPS in control mice and S1 mice. 実施例の結果を示し、対照マウス及びＳ１マウスにおけるコリン作動性ニューロンの障害を示す図である。FIG. 1 shows the results of the Examples and shows the damage of cholinergic neurons in control and S1 mice. 実施例の結果を示し、対照マウス及びＳ１マウスにおけるコリン作動性ニューロンの障害を示す図である。FIG. 1 shows the results of the Examples and shows the damage of cholinergic neurons in control and S1 mice. ドネペジルの投与スキームを説明するための図である。FIG. 3 is a diagram for explaining the administration scheme of donepezil. 実施例の結果を示し、対照マウス及びＳ１マウスにおけるドネペジルの投与後の１０％の重り付き強制水泳試験の結果を示す図である。FIG. 2 shows the results of the Examples, showing the results of the 10% weighted forced swim test after administration of donepezil in control and S1 mice. 実施例の結果を示し、対照マウス及びＳ１マウスにおけるドネペジルの投与後の尾懸垂試験の結果を示す図である。FIG. 4 shows the results of the Examples and shows the results of the tail suspension test after administration of donepezil in control and S1 mice. 実施例の結果を示し、対照マウス及びＳ１マウスにおけるドネペジルの投与後のインターロイキン６（ＩＬ－６）、腫瘍壊死因子（ＴＮＦα）、ケモカインＣＣモチーフリガンド２（ＣＣＬ２）の遺伝子発現量を解析した結果を示す図である。The results of Examples are shown, and the results of analyzing the gene expression levels of interleukin 6 (IL-6), tumor necrosis factor (TNFα), and chemokine CC motif ligand 2 (CCL2) after administration of donepezil in control mice and S1 mice. It is a figure which shows. 実施例の結果を示し、対照マウス及びＳ１マウスにおけるＰＮＵ２８２９８７投与後のインターロイキン１ベータ（ＩＬ－１β）及びインターロイキン６（ＩＬ－６）の遺伝子発現量を解析した結果を示す図である。FIG. 10 shows the results of Examples, and shows the results of analyzing the gene expression levels of interleukin-1 beta (IL-1β) and interleukin-6 (IL-6) after administration of PNU282987 in control mice and S1 mice. 実施例の結果を示し、対照マウス及びＳ１マウスにおけるＰＮＵ２８２９８７投与後のＺＦＰ３６の遺伝子発現量を解析した結果を示す図である。FIG. 10 shows the results of Examples, and shows the results of analyzing the gene expression level of ZFP36 after administration of PNU282987 in control mice and S1 mice.
　＜１．治療薬＞
　（特徴）
　本発明の一態様に係る新型コロナウイルス感染症（ＣＯＶＩＤ－１９）後遺症（以下、「ＣＯＶＩＤ－１９後遺症」）の治療薬は、ＣＯＶＩＤ－１９後遺症の治療又は予防用途に用いられる医薬組成物であり、アセチルコリン受容体作動薬を有効成分として含む。これにより、新型コロナウイルス感染患者においてＣＯＶＩＤ－１９後遺症を治療又は予防することができる。従って、持続可能な開発目標（ＳＤＧｓ）の目標３「すべての人に健康と福祉を」に貢献できる。以下、説明の便宜上、本発明の一態様に係るＣＯＶＩＤ－１９後遺症の治療薬を、単に「治療薬」ということがある。 <1. Therapeutic drugs>
(feature)
A therapeutic drug for the aftereffects of novel coronavirus infection (COVID-19) according to one aspect of the present invention (hereinafter, "COVID-19 aftereffects") is a pharmaceutical composition used for treatment or prevention of COVID-19 aftereffects. , containing an acetylcholine receptor agonist as an active ingredient. This can treat or prevent the sequelae of COVID-19 in patients infected with the novel coronavirus. Therefore, it can contribute to Goal 3 of the Sustainable Development Goals (SDGs) "Good health and well-being for all". Hereinafter, for convenience of explanation, the therapeutic drug for the aftereffects of COVID-19 according to one aspect of the present invention may be simply referred to as "therapeutic drug".
　本明細書において、「治療」とは、本発明の一態様に係る治療薬が投与された対象（以下、単に「投与対象」と称する。）においてＣＯＶＩＤ－１９後遺症の症状を完治又は軽減させること、ＣＯＶＩＤ－１９後遺症の症状の悪化を抑制することを含む。本発明において、「予防」とは、投与対象においてＣＯＶＩＤ－１９後遺症の症状の発症を抑制すること、又は遅延させることを含む。 As used herein, the term "treatment" refers to complete cure or alleviation of symptoms of COVID-19 sequelae in a subject to whom the therapeutic agent according to one aspect of the present invention is administered (hereinafter simply referred to as "administration subject"). , including reducing the exacerbation of symptoms of COVID-19 sequelae. In the present invention, "prevention" includes suppressing or delaying the onset of symptoms of COVID-19 aftereffects in a subject.
　投与対象がＣＯＶＩＤ－１９後遺症の症状を発症している場合、本発明の一態様に係る治療薬は、ＣＯＶＩＤ－１９後遺症の治療用途に用いられる。投与対象がＣＯＶＩＤ－１９後遺症の症状を生じていない場合、本発明の一態様に係る治療薬は、ＣＯＶＩＤ－１９後遺症の予防用途に用いられる。 When the administration subject develops symptoms of the aftereffects of COVID-19, the therapeutic agent according to one aspect of the present invention is used for the treatment of the aftereffects of COVID-19. If the administration subject does not develop symptoms of the aftereffects of COVID-19, the therapeutic agent according to one aspect of the present invention is used for prevention of the aftereffects of COVID-19.
　本明細書において、「治療」及び「予防」の定義は前述のとおりであるが、いずれの場合も本発明の一態様に係る治療薬の作用機序は同じである。従って、「治療薬」は、「予防薬」に置換可能である。すなわち、新型コロナウイルス感染患者がＣＯＶＩＤ－１９後遺症の症状を生じていない場合、「治療」は「予防」を意味する。 In the present specification, the definitions of "treatment" and "prevention" are as described above, and in both cases the mechanism of action of the therapeutic agent according to one aspect of the present invention is the same. Therefore, "therapeutic agent" can be replaced with "prophylactic agent." In other words, 'treatment' means 'prevention' if the novel coronavirus-infected patient does not develop symptoms of COVID-19 sequelae.
　ここで、一般的にＣＯＶＩＤ－１９後遺症は、新型コロナウイルス感染後の回復期に出現し、その後数週間から数カ月にわたって持続する症状全般を意味するがその定義は定まっていない。また、ＣＯＶＩＤ－１９後遺症はＬｏｎｇ　ＣＯＶＩＤと呼ばれることがある。本明細書における「ＣＯＶＩＤ－１９後遺症」は上記の症状のなかでも特に出現頻度の高い疲労、うつ症状、嗅覚障害、記憶障害、集中力の低下、思考力の低下、認知機能の低下といった脳や神経の機能異常が関係する症状を意味する。 Here, the aftereffects of COVID-19 generally refer to symptoms in general that appear during the recovery period after infection with the new coronavirus and persist for several weeks to several months afterward, but the definition is not established. In addition, the aftereffects of COVID-19 are sometimes called Long COVID. "COVID-19 sequelae" in the present specification are fatigue, depressive symptoms, olfactory disorders, memory impairment, decreased concentration, decreased thinking ability, decreased cognitive function, etc., which are particularly frequent among the above symptoms. Refers to symptoms associated with nerve dysfunction.
　本明細書における「ＣＯＶＩＤ－１９後遺症」の一態様は、新型コロナウイルスが関与する疲労である。また、本明細書における「ＣＯＶＩＤ－１９後遺症」の一態様は、新型コロナウイルスが関与するうつ症状である。また、本明細書における「ＣＯＶＩＤ－１９後遺症」の一態様は、新型コロナウイルスが関与する嗅覚障害である。また、本明細書における「ＣＯＶＩＤ－１９後遺症」の一態様は、新型コロナウイルスが関与する記憶障害である。 One aspect of "COVID-19 sequelae" in this specification is fatigue associated with the new coronavirus. Also, one aspect of "COVID-19 sequelae" herein is depressive symptoms associated with the novel coronavirus. In addition, one aspect of the "COVID-19 sequelae" in this specification is olfactory disturbance associated with the novel coronavirus. Also, one aspect of "COVID-19 sequelae" herein is memory impairment associated with the novel coronavirus.
　本明細書において、「新型コロナウイルスが関与するうつ症状」とは、一般的にうつ病と診断される疾患の症状を指す。症状としては、抑うつ気分、興味関心・喜びの感情の喪失、食欲不振、過食、睡眠障害、過眠、精神運動性の焦燥又は制止、易疲労性、無価値感や罪責感、集中力の低下や思考力の低下、自殺念慮などのＤＳＭ－５による大うつ病の診断に利用される症状に加え、不安、記憶力低下、老化、疼痛、慢性疼痛などの、うつ病患者に高頻度でみられる症状を呈するうつ状態であることを意図する。すなわち、本明細書における「新型コロナウイルスが関与するうつ症状」とは、大うつ病に限らず、ストレス性うつ病、双極性障害のうつ状態、否定形うつ病など、上記のうつ症状を示す疾患及び他の疾患におけるうつ状態における症状を含む概念である。 As used herein, "depressive symptoms associated with the new coronavirus" refer to symptoms of diseases that are generally diagnosed as depression. Symptoms include depressed mood, loss of interest and pleasure, anorexia, overeating, sleep disturbance, hypersomnia, psychomotor agitation or inhibition, fatigability, feelings of worthlessness and guilt, and poor concentration. In addition to symptoms used in the diagnosis of major depression according to the DSM-5, such as slowed thinking and suicidal ideation, anxiety, memory loss, aging, pain, chronic pain, etc., are frequently seen in depressed patients. Intended to be symptomatic depression. That is, the "depressive symptoms associated with the novel coronavirus" in the present specification are not limited to major depression, and include the above depressive symptoms such as stress depression, bipolar disorder depression, and negative depression. It is a concept that includes symptoms in depression in disease and other diseases.
　本明細書における「新型コロナウイルスが関与する疲労」とは、中枢神経系などの疾患を伴う持続的あるいは慢性的な現象で、興味関心・喜びの感情の喪失、睡眠障害、精神運動性の焦燥又は制止、易疲労性、集中力の低下や思考力の低下を主な症状とする病的疲労を意味する。「疲労」は、疲労感又は倦怠感と表現することもできる。 As used herein, "fatigue associated with the novel coronavirus" is a persistent or chronic phenomenon associated with diseases of the central nervous system, such as loss of interest and pleasure, sleep disturbance, and psychomotor agitation. Or it means pathological fatigue whose main symptoms are retardation, fatigability, poor concentration and poor thinking. "Fatigue" can also be expressed as tiredness or malaise.
　本明細書における「病的疲労」とは、新型コロナウイルス感染症に伴う持続的あるいは慢性的な疲労を意味する。健康な人が、身体的あるいは精神的負荷を連続して与えられたときにみられる一時的な身体的及び精神的作業能力の質的あるいは量的な低下現象を意味する「生理的疲労」とは区別される。 "Pathological fatigue" as used herein means persistent or chronic fatigue associated with the novel coronavirus infection. "Physiological fatigue" refers to the phenomenon of temporary qualitative or quantitative decline in physical and mental work capacity seen when a healthy person is subjected to a continuous physical or mental load. are distinguished.
　本明細書における「新型コロナウイルスが関与する嗅覚障害」とは、嗅覚つまり「におい」の感覚に何らかの異常を来す症状又は疾患である。「嗅覚異常」ともいう。主な症状としては、嗅覚減衰症、嗅覚錯誤、無嗅覚症、嗅覚過敏が挙げられる。 In this specification, "olfactory disorders associated with the new coronavirus" are symptoms or diseases that cause some kind of abnormality in the sense of smell, that is, the sense of "smell". Also known as olfactory abnormality. The main symptoms include anosmia, olfactory illusion, anosmia, and hyperosmia.
　本明細書における「新型コロナウイルスが関与する記憶障害」とは、記憶を構成する記銘、保持、再生及び再認の４つの過程のいずれか又は全てが正常に働かない状態を意味する。 As used herein, "memory impairment associated with the novel coronavirus" means a state in which any or all of the four processes that make up memory - recording, retention, reproduction, and recognition - do not function properly.
　なお、本明細書における「症状」とは、新型コロナウイルスが関与する疾患の影響により患者に発現する現象及び状態（異常）を意味し、患者本人が、該疾患の症状であることを知覚できる異常である自覚症状と、医師の診察又は検査等によって、該疾患の症状であることを客観的に確かめられる異常である他覚症状と、を含む概念である。尚、後述する動物モデルにおいても行動の変化と検査などによる他覚所見を合わせて症状と呼ぶ。 As used herein, the term "symptoms" means phenomena and conditions (abnormalities) that occur in patients due to the effects of diseases associated with the novel coronavirus, and the patients themselves can perceive that they are symptoms of the disease. It is a concept that includes subjective symptoms that are abnormal and objective symptoms that are abnormal and can be objectively confirmed to be symptoms of the disease by medical examination or examination by a doctor. In the animal model described later, changes in behavior and objective findings from examinations are collectively referred to as "symptoms."
　本発明者らは、ＣＯＶＩＤ－１９後遺症モデル動物を用いたＣＯＶＩＤ－１９後遺症の脳変化を研究した。この結果、ＣＯＶＩＤ－１９後遺症の脳では、前脳基底部の中隔野（ＭＳ）及びブローカの対角帯（ＤＢ）のコリンアセチルトランスフェラーゼ（ＣｈＡＴ）陽性細胞であるコリン作動性ニューロンの数が低下していることを初めて見出した。 The inventors studied brain changes in COVID-19 sequelae using model animals of COVID-19 sequelae. This resulted in reduced numbers of cholinergic neurons, choline acetyltransferase (ChAT)-positive cells, in the basal forebrain septal area (MS) and Broca's diagonal zone (DB) in brains with COVID-19 sequelae. I discovered for the first time that
　この結果は、ＣＯＶＩＤ－１９後遺症ではアセチルコリン量が減少していることを示唆するものであり、アセチルコリン受容体作動薬がＣＯＶＩＤ－１９後遺症の治療薬として相応しいことを示唆している。 This result suggests that the amount of acetylcholine is reduced in the aftereffects of COVID-19, suggesting that acetylcholine receptor agonists are suitable as therapeutic agents for the aftereffects of COVID-19.
　本発明者らは、ＣＯＶＩＤ－１９後遺症モデル動物を用いたＣＯＶＩＤ－１９後遺症の研究の結果、アセチルコリン受容体作動薬を投与することで、ＣＯＶＩＤ－１９後遺症モデル動物において発現しているうつ症状及び疲労の症状が改善することを初めて見出した。つまり、アセチルコリン受容体作動薬を投与することで、ＣＯＶＩＤ－１９後遺症を治療又は予防できることを初めて見出した。 As a result of research on COVID-19 sequelae using COVID-19 sequelae model animals, the present inventors found that by administering an acetylcholine receptor agonist, depressive symptoms and fatigue expressed in COVID-19 sequelae model animals It was found for the first time that the symptoms of In other words, the present inventors have found for the first time that the aftereffects of COVID-19 can be treated or prevented by administering an acetylcholine receptor agonist.
　従来、アセチルコリン受容体作動薬の投与によって脳内のアセチルコリン量が上昇すると、うつ症状が発症することが技術常識であった（例えば、参考文献１：Cholinergic regulation of mood: from basic and clinical studies to emerging therapeutics. Stephanie C Dulawa and David S Janowsky. Mol Psychiatry. 2019 May;24(5):694-709.及び参考文献２：Maintenance treatment of depression in old age: a randomized, double-blind, placebo-controlled evaluation of the efficacy and safety of donepezil combined with antidepressant pharmacotherapy. Charles F Reynolds 3rd, Meryl A Butters, Oscar Lopez. et.al. Arch Gen Psychiatry. 2011;68(1):51-60.）。 Conventionally, it was common general knowledge that depressive symptoms develop when the amount of acetylcholine in the brain increases due to the administration of acetylcholine receptor agonists (for example, Reference 1: Cholinergic regulation of mood: from basic and clinical studies to emerging therapeutics. Stephanie C Dulawa and David S Janowsky. Mol Psychiatry. 2019 May;24(5):694-709. the efficiency and safety of donepezil combined with antidepressant pharmacotherapy. Charles F Reynolds 3rd, Meryl A Butters, Oscar Lopez. et al. Arch Gen Psychiatry. 2011;68(1):51-60.).
　また、喫煙が新型コロナウイルスの症状を進行・悪化させることや、その原因がニコチンにあることが技術常識であった（例えば、参考文献３：Tobacco smoking and COVID-19 infection. Richard N van Zyl-Smit, Guy Richards, Frank T Leone. Lancet Respir Med. 2020 Jul;8(7):664-665.；参考文献４：Smoking Is Associated With COVID-19 Progression: A Meta-analysis. Roengrudee Patanavanich, Stanton A Glantz. Nicotine Tob Res. 2020 Aug 24;22(9):1653-1656.；参考文献５：The Role of Smoking and Nicotine in the Transmission and Pathogenesis of COVID-19. Ali Ehsan Sifat, Saeideh Nozohouri, Heidi Villalba, Bhuvaneshwar Vaidya, Thomas J Abbruscato J Pharmacol Exp Ther. 2020 Dec;375(3):498-509.；参考文献６：The Effect of Smoking on COVID-19 Symptom Severity: Systematic Review and Meta-Analysis Askin Gulsen 1, Burcu Arpinar Yigitbas 2, Berat Uslu 2, Daniel Dromann 1, Oguz Kilinc Pulm Med. 2020 Sep 8;2020:7590207.）。 In addition, it was common technical knowledge that smoking worsens the symptoms of the new coronavirus and that nicotine is the cause (for example, reference 3: Tobacco smoking and COVID-19 infection. Richard N van Zyl- Smit, Guy Richards, Frank T Leone. Lancet Respir Med. 2020 Jul;8(7):664-665.; Reference 4: Smoking Is Associated With COVID-19 Progression: A Meta-analysis. Roengrudee Patanavanich, Stanton A Glantz 2020 Aug 24;22(9):1653-1656.; Reference 5: The Role of Smoking and Nicotine in the Transmission and Pathogenesis of COVID-19. Ali Ehsan Sifat, Saeideh Nozohouri, Heidi Villalba, Bhuvaneshwar Vaidya, Thomas J Abbruscato J Pharmacol Exp Ther. 2020 Dec;375(3):498-509.; Reference 6: The Effect of Smoking on COVID-19 Symptom Severity: Systematic Review and Meta-Analysis Askin Gulsen 1, Burcu Arpinar. Yigitbas 2, Berat Uslu 2, Daniel Dromann 1, Oguz Kilinc Pulm Med. 2020 Sep 8;2020:7590207.).
　これに対して、アセチルコリン受容体作動薬の投与によって脳内のアセチルコリン量を増加させることで、ＣＯＶＩＤ－１９後遺症モデル動物において発現している新型コロナウイルスが関与するうつ症状を改善し得るという結果は、従来の技術常識からアセチルコリン受容体作動薬の効果として予想される結果とは全く逆の結果であるといえる。また、この結果から、ＣＯＶＩＤ－１９後遺症によるうつ症状（つまり、「新型コロナウイルスが関与するうつ症状」）は、アセチルコリン量の上昇に起因するうつ病とは異なる発症機構によって発症すると考えられる。 On the other hand, the results show that increasing the amount of acetylcholine in the brain by administering an acetylcholine receptor agonist can improve the depressive symptoms associated with the new coronavirus expressed in model animals with the aftereffects of COVID-19. , it can be said that the result is completely opposite to the result expected as an effect of an acetylcholine receptor agonist from the conventional technical common sense. In addition, from this result, it is considered that depressive symptoms due to the aftereffects of COVID-19 (that is, "depressive symptoms associated with the new coronavirus") develop by a different onset mechanism from depression caused by an increase in the amount of acetylcholine.
　（有効成分）
　本発明の一態様に係る治療薬は、アセチルコリン受容体作動薬を有効成分として含む。本明細書において、「アセチルコリン受容体作動薬」とは、コリンエステラーゼ阻害作用などによって、脳内のアセチルコリンの量を増加させる間接型のアセチルコリン受容体作動薬と、受容体に直接結合して作用する直接型のアセチルコリン受容体作動薬の両者を意図する。本発明の一態様において、アセチルコリン受容体作動薬は、副交感神経などを介して作用する末梢性のアセチルコリン受容体作動薬であってもよく、脳内の受容体へ作用する中枢性のアセチルコリン受容体作動薬であってもよい。前述のとおり、ＣＯＶＩＤ－１９後遺症は脳や神経の機能異常が関係する症状であり、機能異常が生じている部位に直接作用できるという利点があることから、アセチルコリン受容体作動薬は、脳内のアセチルコリン受容体に作用する中枢性のアセチルコリン受容体作動薬であることが好ましい。「アセチルコリン受容体作動薬」は、コリン作動薬とも称される。 (active ingredient)
A therapeutic agent according to one aspect of the present invention contains an acetylcholine receptor agonist as an active ingredient. As used herein, the term "acetylcholine receptor agonist" refers to an indirect acetylcholine receptor agonist that increases the amount of acetylcholine in the brain by cholinesterase inhibitory action or the like, and a direct acetylcholine receptor agonist that acts by directly binding to the receptor. Both types of acetylcholine receptor agonists are contemplated. In one aspect of the present invention, the acetylcholine receptor agonist may be a peripheral acetylcholine receptor agonist that acts via parasympathetic nerves or the like, or a central acetylcholine receptor agonist that acts on receptors in the brain. It may be an agonist. As mentioned above, the aftereffects of COVID-19 are symptoms related to brain and nerve dysfunction. It is preferably a central acetylcholine receptor agonist that acts on acetylcholine receptors. "Acetylcholine receptor agonists" are also referred to as cholinergics.
　中枢性のアセチルコリン受容体作動薬が作用する脳内の受容体としては、より具体的には、嗅球、海馬、中隔野、及び／又は、嗅結節等が挙げられるが、アセチルコリン受容体作動薬が作用する箇所は多数存在することが知られており、ここに例示したものに限定されるわけではない。 Receptors in the brain on which central acetylcholine receptor agonists act include, more specifically, the olfactory bulb, hippocampus, septal area, and/or olfactory tubercle. It is known that there are a number of locations on which is acted, and it is not limited to those exemplified here.
　直接型のアセチルコリン受容体作動薬としては、血液脳関門を横断して脳に到達可能であり、かつ、アセチルコリン受容体を活性化する作用を有する物質であることが好ましい。直接型のアセチルコリン受容体作動薬としては、より具体的には、アセチルコリン及びその前駆体、ならびに、アセチルコリン受容体（ムスカリン受容体又はニコチン受容体）のアゴニスト等が挙げられる。 A direct-type acetylcholine receptor agonist is preferably a substance that can cross the blood-brain barrier to reach the brain and has the effect of activating the acetylcholine receptor. More specific examples of direct acetylcholine receptor agonists include acetylcholine and its precursors, and agonists of acetylcholine receptors (muscarinic receptors or nicotinic receptors).
　間接型のアセチルコリン受容体作動薬としては、血液脳関門を横断して脳に到達可能であり、かつ、アセチルコリンエステラーゼ阻害作用を有する物質であることが好ましい。間接型のアセチルコリン受容体作動薬としては、より具体的には、ドネペジル（２－［（１－ベンジル－４－ピペリジニル）メチル］－５，６－ジメトキシインダン－１－オン塩酸塩）；リバスティグミン（２，６－ジオキソ－４－フェニル－ピペリジン－３－カルボニトリル）；メトリフォネート（Ｏ，Ｏ－ジメチル－２，２，２－トリクロロ－１－ヒドロキシエチルホスホナートエステル）；タクリン（１，２，３，４－テトラヒドロ－９－アミノアクリジン）；ガランタミン（ガランタミン臭化水素酸塩）等が挙げられる。 The indirect acetylcholine receptor agonist is preferably a substance that can cross the blood-brain barrier to reach the brain and has an acetylcholinesterase inhibitory action. Indirect acetylcholine receptor agonists, more specifically, donepezil (2-[(1-benzyl-4-piperidinyl)methyl]-5,6-dimethoxyindan-1-one hydrochloride); amine (2,6-dioxo-4-phenyl-piperidine-3-carbonitrile); metrifonate (O,O-dimethyl-2,2,2-trichloro-1-hydroxyethylphosphonate ester); tacrine (1 , 2,3,4-tetrahydro-9-aminoacridine); galantamine (galantamine hydrobromide);
　また、例示したアセチルコリン受容体作動薬の中でも、ドネペジルは、すでにアルツハイマー病の治療薬等として使用されているため、ドラッグリポジショニングが可能である。それゆえ、（ｉ）ヒトでの安全性試験、薬物動態試験を短縮又は省略できる、（ｉｉ）副作用が既知であるため、患者の負担の少ない有効成分を選択可能である、（ｉｉｉ）有効成分の製造方法がすでに確立していることから薬価を低く抑えることができる、等の利点を有する。したがって、本発明の一態様において、アセチルコリン受容体作動薬は、ドネペジルであることが好ましい。 In addition, among the acetylcholine receptor agonists exemplified, donepezil is already used as a therapeutic drug for Alzheimer's disease, etc., so drug repositioning is possible. Therefore, (i) human safety tests and pharmacokinetic tests can be shortened or omitted, (ii) side effects are known, so an active ingredient with less burden on the patient can be selected, (iii) active ingredient It has the advantage of being able to keep the drug price low because the manufacturing method has already been established. Therefore, in one aspect of the present invention, the acetylcholine receptor agonist is preferably donepezil.
　なお、アセチルコリン受容体作動薬は、脳内のアセチルコリンの量を増加させる作用を有する限り、「誘導体」の形態であってもよく、「薬理学的に許容され得る塩」の形態であってもよい。すなわち、本明細書において、「アセチルコリン受容体作動薬」とは、その「誘導体」、ならびに、「薬理学的に許容され得る塩」も含む概念である。 The acetylcholine receptor agonist may be in the form of a "derivative" or a "pharmacologically acceptable salt" as long as it has the effect of increasing the amount of acetylcholine in the brain. good. That is, in the present specification, the term "acetylcholine receptor agonist" is a concept including its "derivatives" and "pharmacologically acceptable salts".
　本明細書において、「誘導体」とは、特定の化合物に対して、該化合物の分子内の一部が、他の官能基又は他の原子と置換されることにより生じる化合物群を意図する。上記他の官能基の例としては、アルキル基、アルコキシ基、アルキルチオ基、アリール基、アリールオキシ基、アリールチオ基、アリールアルキル基、アリールアルコキシ基、アリールアルキルチオ基、アリールアルケニル基、アリールアルキニル基、アリル基、アミノ基、置換アミノ基、シリル基、置換シリル基、シリルオキシ基、置換シリルオキシ基、アリールスルフォニルオキシ基、アルキルスルフォニルオキシ基、ニトロ基などが挙げられる。上記他の原子の例としては、炭素原子、水素原子、酸素原子、窒素原子、硫黄原子、リン原子、ハロゲン原子などが挙げられる。 As used herein, the term "derivative" refers to a group of compounds produced by substituting a part of the molecule of a specific compound with another functional group or another atom. Examples of other functional groups include alkyl groups, alkoxy groups, alkylthio groups, aryl groups, aryloxy groups, arylthio groups, arylalkyl groups, arylalkoxy groups, arylalkylthio groups, arylalkenyl groups, arylalkynyl groups, and allyl. groups, amino groups, substituted amino groups, silyl groups, substituted silyl groups, silyloxy groups, substituted silyloxy groups, arylsulfonyloxy groups, alkylsulfonyloxy groups, nitro groups and the like. Examples of the other atoms include carbon atoms, hydrogen atoms, oxygen atoms, nitrogen atoms, sulfur atoms, phosphorus atoms, halogen atoms and the like.
　誘導体としては、生体内（インビボな条件）等において、加水分解、酸化、あるいは、酵素反応等により、所望の活性を発揮するプロドラッグを使用することもできる。 As a derivative, it is also possible to use a prodrug that exhibits the desired activity by hydrolysis, oxidation, enzymatic reaction, or the like in vivo (under in vivo conditions).
　本明細書において、「薬学的に許容される塩」とは、医薬品として被験体に投与することが生理学的に許容されうる塩を意図し、その具体例は限定されない。塩の例としては、アルカリ金属塩（カリウム塩など）、アルカリ土類金属塩（カルシウム塩、マグネシウム塩など）、アンモニウム塩、有機塩基塩（トリメチルアミン塩、トリエチルアミン塩、ピリジン塩、ピコリン塩、ジシクロヘキシルアミン塩、Ｎ，Ｎ’－ジベンジルエチレンジアミン塩など）、有機酸塩（酢酸塩、マレイン酸塩、酒石酸塩、メタンスルホン酸塩、ベンゼンスルホン酸塩、蟻酸塩、トルエンスルホン酸塩、トリフルオロ酢酸塩など）、無機酸塩（塩酸塩、臭化水素酸塩、硫酸塩、燐酸塩など）を挙げられる。 As used herein, the term "pharmaceutically acceptable salt" intends a salt that is physiologically acceptable for administration to a subject as a pharmaceutical, and specific examples thereof are not limited. Examples of salts include alkali metal salts (potassium salts, etc.), alkaline earth metal salts (calcium salts, magnesium salts, etc.), ammonium salts, organic base salts (trimethylamine salts, triethylamine salts, pyridine salts, picoline salts, dicyclohexylamine salt, N,N'-dibenzylethylenediamine salt, etc.), organic acid salt (acetate, maleate, tartrate, methanesulfonate, benzenesulfonate, formate, toluenesulfonate, trifluoroacetate) etc.), inorganic acid salts (hydrochlorides, hydrobromides, sulfates, phosphates, etc.).
　本発明の一態様において、アセチルコリン受容体作動薬は、アセチルコリン受容体以外の受容体を活性化、又は、阻害してもよいが、意図しない副作用等を抑制する観点から、アセチルコリン受容体を選択的（特異的）に活性化する化合物であることが好ましい。 In one aspect of the present invention, the acetylcholine receptor agonist may activate or inhibit receptors other than the acetylcholine receptor, but from the viewpoint of suppressing unintended side effects, etc., the acetylcholine receptor agonist is selectively used. It is preferably a (specifically) activating compound.
　（その他の成分）
　本発明の一態様に係る治療薬は、前述した有効成分（アセチルコリン受容体作動薬）以外の成分を含有していてもよい。有効成分以外の成分は、薬学的に許容され得る成分であればよく、例えば、緩衝剤、ｐＨ調整剤、等張化剤、防腐剤、抗酸化剤、高分子量重合体、賦形剤、溶媒などであり得る。 (other ingredients)
The therapeutic agent according to one aspect of the present invention may contain ingredients other than the active ingredient (acetylcholine receptor agonist) described above. Ingredients other than the active ingredient may be pharmaceutically acceptable ingredients, such as buffers, pH adjusters, tonicity agents, preservatives, antioxidants, high molecular weight polymers, excipients, solvents and so on.
　前記緩衝剤の例としては、リン酸又はリン酸塩、ホウ酸又はホウ酸塩、クエン酸又はクエン酸塩、酢酸又は酢酸塩、炭酸又は炭酸塩、酒石酸又は酒石酸塩、ε－アミノカプロン酸、トロメタモールなどが挙げられる。前記リン酸塩としては、リン酸ナトリウム、リン酸二水素ナトリウム、リン酸水素二ナトリウム、リン酸カリウム、リン酸二水素カリウム、リン酸水素二カリウムなどが挙げられる。前記ホウ酸塩としては、ホウ砂、ホウ酸ナトリウム、ホウ酸カリウムなどが挙げられる。前記クエン酸塩としては、クエン酸ナトリウム、クエン酸二ナトリウム、クエン酸三ナトリウムなどが挙げられる。前記酢酸塩としては、酢酸ナトリウム、酢酸カリウムなどが挙げられる。前記炭酸塩としては、炭酸ナトリウム、炭酸水素ナトリウムなどが挙げられる。前記酒石酸塩としては、酒石酸ナトリウム、酒石酸カリウムなどが挙げられる。 Examples of said buffers include phosphoric acid or phosphate, boric acid or borate, citric acid or citrate, acetic acid or acetate, carbonic acid or carbonate, tartaric acid or tartrate, ε-aminocaproic acid, trometamol etc. Examples of the phosphate include sodium phosphate, sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium phosphate, potassium dihydrogen phosphate, and dipotassium hydrogen phosphate. Examples of the borate include borax, sodium borate, and potassium borate. Examples of the citrate include sodium citrate, disodium citrate, and trisodium citrate. Examples of the acetate include sodium acetate and potassium acetate. Examples of the carbonate include sodium carbonate and sodium hydrogen carbonate. Examples of the tartrate include sodium tartrate and potassium tartrate.
　前記ｐＨ調整剤の例としては、塩酸、リン酸、クエン酸、酢酸、水酸化ナトリウム、水酸化カリウムなどが挙げられる。 Examples of the pH adjuster include hydrochloric acid, phosphoric acid, citric acid, acetic acid, sodium hydroxide, potassium hydroxide, and the like.
　前記等張化剤の例としては、イオン性等張化剤（塩化ナトリウム、塩化カリウム、塩化カルシウム、塩化マグネシウムなど）、非イオン性等張化剤（グリセリン、プロピレングリコール、ソルビトール、マンニトールなど）が挙げられる。 Examples of the tonicity agents include ionic tonicity agents (sodium chloride, potassium chloride, calcium chloride, magnesium chloride, etc.) and nonionic tonicity agents (glycerin, propylene glycol, sorbitol, mannitol, etc.). mentioned.
　前記防腐剤の例としては、ベンザルコニウム塩化物、ベンザルコニウム臭化物、ベンゼトニウム塩化物、ソルビン酸、ソルビン酸カリウム、パラオキシ安息香酸メチル、パラオキシ安息香酸プロピル、クロロブタノールなどが挙げられる。 Examples of the antiseptic include benzalkonium chloride, benzalkonium bromide, benzethonium chloride, sorbic acid, potassium sorbate, methyl parahydroxybenzoate, propyl parahydroxybenzoate, and chlorobutanol.
　前記抗酸化剤の例としては、アスコルビン酸、トコフェノール、ジブチルヒドロキシトルエン、ブチルヒドロキシアニソール、エリソルビン酸ナトリウム、没食子酸プロピル、亜硫酸ナトリウムなどが挙げられる。 Examples of the antioxidant include ascorbic acid, tocopherol, dibutylhydroxytoluene, butylhydroxyanisole, sodium erythorbate, propyl gallate, and sodium sulfite.
　前記高分子量重合体の例としては、メチルセルロース、エチルセルロース、ヒドロキシメチルセルロース、ヒドロキシエチルセルロース、ヒドロキシプロピルセルロース、ヒドロキシエチルメチルセルロース、ヒドロキシプロピルメチルセルロース、カルボキシメチルセルロース、カルボキシメチルセルロースナトリウム、ヒドロキシプロピルメチルセルロースアセテートサクシネート、ヒドロキシプロピルメチルセルロースフタレート、カルボキシメチルエチルセルロース、酢酸フタル酸セルロース、ポリビニルピロリドン、ポリビニルアルコール、カルボキシビニルポリマー、ポリエチレングリコール、アテロコラーゲンなどが挙げられる。 Examples of the high molecular weight polymers include methylcellulose, ethylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxyethylmethylcellulose, hydroxypropylmethylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, hydroxypropylmethylcellulose acetate succinate, hydroxypropylmethylcellulose phthalate. , carboxymethylethylcellulose, cellulose acetate phthalate, polyvinylpyrrolidone, polyvinyl alcohol, carboxyvinyl polymer, polyethylene glycol, atelocollagen, and the like.
　前記賦形剤の例としては、乳糖、白糖、Ｄ－マンニトール、キシリトール、ソルビトール、エリスリトール、デンプン、結晶セルロースなどが挙げる。 Examples of the excipient include lactose, sucrose, D-mannitol, xylitol, sorbitol, erythritol, starch, and crystalline cellulose.
　前記溶媒の例としては、水、生理的食塩水、アルコールなどが挙げられる。 Examples of the solvent include water, physiological saline, and alcohol.
　本発明の一態様に係る治療薬は、前述した他の成分として所望の効果（例えば、副作用の低減）を有する薬効成分を含んでいてもよく、所望の効果を有する薬剤と併用されてもよい。 The therapeutic agent according to one aspect of the present invention may contain a medicinal ingredient having the desired effect (e.g., reduction of side effects) as the other component described above, and may be used in combination with a drug having the desired effect. .
　（有効成分及びその他の成分の含有量）
　本発明の一態様に係る治療薬中の有効成分の量は、特に限定されない。有効成分の量は、例えば、本発明の一態様に係る治療薬の総重量に対して、０．００１重量％～１００重量％であってもよく、０．０１重量％～１００重量％であってもよく、０．１重量％～１００重量％であってもよく、０．１重量％～９５重量％であってもよく、０．１重量％～９０重量％であってもよく、０．１重量％～８０重量％であってもよく、０．１重量％～７０重量％であってもよく、０．１重量％～６０重量％であってもよく、０．１重量％～５０重量％であってもよく、０．１重量％～４０重量％であってもよく、０．１重量％～３０重量％であってもよく、０．１重量％～２０重量％であってもよく、０．１重量％～１０重量％であってもよい。 (Content of active ingredients and other ingredients)
The amount of active ingredient in the therapeutic agent according to one aspect of the present invention is not particularly limited. The amount of active ingredient may be, for example, 0.001% to 100% by weight, or 0.01% to 100% by weight, relative to the total weight of the therapeutic agent according to one aspect of the present invention. 0.1 wt% to 100 wt%, 0.1 wt% to 95 wt%, 0.1 wt% to 90 wt%, 0 .1 wt% to 80 wt%, 0.1 wt% to 70 wt%, 0.1 wt% to 60 wt%, 0.1 wt% to It may be 50% by weight, 0.1% to 40% by weight, 0.1% to 30% by weight, or 0.1% to 20% by weight. may be from 0.1% to 10% by weight.
　本発明の一態様に係る治療薬中の有効成分以外の成分の量は、特に限定されない。有効成分以外の成分の量は、例えば、本発明の一態様に係る治療薬の総重量に対して、０重量％～９９．９９９重量％であってもよく、０重量％～９９．９９重量％であってもよく、０重量％～９９．９重量％であってもよく、５重量％～９９．９重量％であってもよく、１０重量％～９９．９重量％であってもよく、２０重量％～９９．９重量％であってもよく、３０重量％～９９．９重量％であってもよく、４０重量％～９９．９重量％であってもよく、５０重量％～９９．９重量％であってもよく、６０重量％～９９．９重量％であってもよく、７０重量％～９９．９重量％であってもよく、８０重量％～９９．９重量％であってもよく、９０重量％～９９．９重量％であってもよい。 The amount of ingredients other than the active ingredient in the therapeutic agent according to one aspect of the present invention is not particularly limited. The amount of ingredients other than the active ingredient may be, for example, 0% to 99.999% by weight, or 0% to 99.99% by weight, relative to the total weight of the therapeutic agent according to one aspect of the present invention. %, may be 0 wt% to 99.9 wt%, may be 5 wt% to 99.9 wt%, or may be 10 wt% to 99.9 wt% well, it may be from 20 wt% to 99.9 wt%, it may be from 30 wt% to 99.9 wt%, it may be from 40 wt% to 99.9 wt%, it may be 50 wt% may be ~99.9 wt%, may be 60 wt% to 99.9 wt%, may be 70 wt% to 99.9 wt%, may be 80 wt% to 99.9 wt% %, or 90% to 99.9% by weight.
　（投与対象）
　本発明の一態様に係る治療薬の投与対象の一例として、新型コロナウイルスの感染が疑われる、又は感染していることが確定した対象が挙げられる。このような投与対象は、コロナウイルス後遺症について医師による診断を受けていない対象であってよい。 (Administration target)
An example of a subject to whom the therapeutic agent according to one aspect of the present invention is administered includes a subject suspected of being infected with, or confirmed to be infected with, the novel coronavirus. Such an administration subject may be a subject who has not been diagnosed by a doctor for coronavirus sequelae.
　本発明の一態様に係る治療薬の投与対象としては、特に限定されず、ヒトであってもよく、非ヒト哺乳動物（例えば、家畜、愛玩動物、及び実験動物）であってもよい。非ヒト哺乳動物としては、例えば、サル、チンパンジー、ウシ、ブタ、ヒツジ、ヤギ、ウマ、イヌ、ネコ、ウサギ、マウス、及びラットが挙げられる。 The subject of administration of the therapeutic agent according to one aspect of the present invention is not particularly limited, and may be humans or non-human mammals (eg, domestic animals, pets, and experimental animals). Non-human mammals include, for example, monkeys, chimpanzees, cows, pigs, sheep, goats, horses, dogs, cats, rabbits, mice, and rats.
　（投与経路）
　本発明の一態様に係る治療薬は、任意の投与経路によって投与対象に投与され得る。投与経路の例としては、経口投与、非経口投与、経皮投与、経粘膜投与、経静脈投与が挙げられる。したがって、本発明の一態様に係る治療薬の剤形は、内服薬、外用薬、又は注射剤などであり得る。投与が簡単であり、投与対象に対する負担が少ないことから、本発明の一態様に係る治療薬の投与経路は、経口投与であることが好ましい。したがって、本発明の一態様に係る治療薬の剤形は、内服薬であることが好ましい。 (Administration route)
A therapeutic agent according to one aspect of the present invention can be administered to an administration subject by any administration route. Examples of administration routes include oral administration, parenteral administration, transdermal administration, transmucosal administration, and intravenous administration. Therefore, the dosage form of the therapeutic agent according to one aspect of the present invention can be an internal medicine, an external medicine, an injection, or the like. The route of administration of the therapeutic agent according to one aspect of the present invention is preferably oral administration, because administration is simple and the burden on the administration subject is small. Therefore, the dosage form of the therapeutic drug according to one aspect of the present invention is preferably an oral drug.
　（製剤及び処方）
　有効成分であるアセチルコリン受容体作動薬、及びその他の成分を原料として、公知の手法により、本発明の一態様に係る治療薬を製剤することができる。 (Formulation and formulation)
A therapeutic agent according to one aspect of the present invention can be formulated by a known method using an acetylcholine receptor agonist as an active ingredient and other ingredients as raw materials.
　本発明の一態様に係る治療薬を対象へ投与する場合、所望の効果が得られるならば、対象への投与量に制限はない。例えば、本発明の一態様に係る治療薬は、有効成分であるアセチルコリン受容体作動薬の投与量が、０．１ｍｇ～１０００．０ｍｇ／ｋｇ体重となるように投与されてもよく、０．１ｍｇ～５００．０ｍｇ／ｋｇ体重となるように投与されてもよく、１．０ｍｇ～５００．０ｍｇ／ｋｇ体重となるように投与されてもよく、１．０ｍｇ～３００．０ｍｇ／ｋｇ体重となるように投与されてもよく、１．０ｍｇ～１００．０ｍｇ／ｋｇ体重となるように投与されてもよく、１．０ｍｇ～５０．０ｍｇ／ｋｇ体重となるように投与されてもよく、１．０ｍｇ～１０．０ｍｇ／ｋｇとなるように投与されてもよく、１．０～１０．０ｍｇ／ｋｇ体重となるように投与されてもよく、１．０～５．０ｍｇ／ｋｇ体重となるように投与されてもよい。 When administering the therapeutic agent according to one aspect of the present invention to a subject, there is no limitation on the dosage to the subject as long as the desired effect is obtained. For example, the therapeutic agent according to one aspect of the present invention may be administered such that the dose of the active ingredient, the acetylcholine receptor agonist, is 0.1 mg to 1000.0 mg/kg body weight. ~500.0 mg/kg body weight may be administered, 1.0 mg to 500.0 mg/kg body weight may be administered, and 1.0 mg to 300.0 mg/kg body weight may be administered. may be administered to 1.0 mg to 100.0 mg/kg body weight, may be administered to 1.0 mg to 50.0 mg/kg body weight, 1.0 mg may be administered to be ~10.0 mg/kg, may be administered to be 1.0 to 10.0 mg/kg body weight, may be administered to be 1.0 to 5.0 mg/kg body weight may be administered.
　本発明の一態様に係る治療薬を対象へ投与する場合、所望の効果が得られるならば、対象への投与間隔に制限はない。投与間隔は、例えば、１時間に１回、１～６時間に１回、６～１２時間に１回、１２時間～１日あたり１回、１日～３日あたり一回、１日～５日あたり１回、１日～７日あたり１回、７日～１４日あたり１回、１４日～２１日あたり１回、１カ月あたり１回、２カ月あたり１回、３カ月あたり１回、４カ月あたり１回、５カ月あたり１回、６カ月あたり１回、又は、１年あたり１回であり得る。また、投与間隔は、一定であってもよいが、ＣＯＶＩＤ－１９後遺症の症状が強く表れた際に、その都度投与（頓服）されてもよい。 When administering the therapeutic agent according to one aspect of the present invention to a subject, there is no limitation on the administration interval to the subject as long as the desired effect is obtained. The administration interval is, for example, once an hour, once every 1 to 6 hours, once every 6 to 12 hours, once every 12 hours to once a day, once every 1 to 3 days, once every 1 to 5 days. once a day, once every 1-7 days, once every 7-14 days, once every 14-21 days, once a month, once every 2 months, once every 3 months, It can be once every four months, once every five months, once every six months, or once a year. In addition, the administration interval may be constant, but may be administered (abruptly) each time symptoms of the aftereffects of COVID-19 appear strongly.
　本発明の一態様に係る治療薬は、例えば、１日あたり一回、有効成分であるアセチルコリン受容体作動薬の投与量が、１．０～５．０ｍｇ／ｋｇ体重となるように投与されてもよい。 The therapeutic agent according to one aspect of the present invention is administered, for example, once a day so that the dose of the active ingredient, the acetylcholine receptor agonist, is 1.0 to 5.0 mg/kg body weight. good too.
　＜２．薬剤のスクリーニング方法＞
　（特徴）
　本発明の一態様に係るＣＯＶＩＤ－１９後遺症治療薬のスクリーニング方法は、非ヒト哺乳動物にＳＡＲＳ－ＣｏＶ－２　Ｓ１タンパク質を発現させたＣＯＶＩＤ－１９後遺症モデル動物を用いて、ＣＯＶＩＤ－１９後遺症の治療又は予防効果を有するＣＯＶＩＤ－１９後遺症治療薬をスクリーニングする方法であり、該ＣＯＶＩＤ－１９後遺症モデル動物に試験物質を投与する投与工程と、ＣＯＶＩＤ－１９後遺症モデル動物における、試験物質の投与前後の、新型コロナウイルスが関与する症状の変化を評価する評価工程と、を含む。これにより、ＣＯＶＩＤ－１９後遺症治療薬をスクリーニングすることができる。その結果、新たなＣＯＶＩＤ－１９後遺症治療薬の薬剤開発につながる。従って、持続可能な開発目標（ＳＤＧｓ）の目標３「すべての人に健康と福祉を」に貢献できる。以下、説明の便宜上、本発明の一態様に係るＣＯＶＩＤ－１９後遺症治療薬のスクリーニング方法を、単に「薬剤のスクリーニング方法」ということがある。 <2. Drug Screening Method>
(feature)
A screening method for a therapeutic drug for COVID-19 sequelae according to one aspect of the present invention uses a COVID-19 sequelae model animal in which SARS-CoV-2 S1 protein is expressed in a non-human mammal, and treats COVID-19 sequelae. Or a method of screening for a therapeutic drug for COVID-19 sequelae having a preventive effect, wherein an administration step of administering a test substance to the COVID-19 sequelae model animal, and before and after administration of the test substance in the COVID-19 sequelae model animal, and an evaluation step of assessing changes in symptoms associated with the novel coronavirus. This makes it possible to screen drugs for the treatment of COVID-19 sequelae. As a result, it will lead to the development of new drugs to treat the aftereffects of COVID-19. Therefore, it can contribute to Goal 3 of the Sustainable Development Goals (SDGs) "Good health and well-being for all". Hereinafter, for convenience of explanation, the method of screening for a drug for treating the sequelae of COVID-19 according to one aspect of the present invention may be simply referred to as a "method of screening a drug."
　（ＣＯＶＩＤ－１９後遺症モデル動物）
　本発明の一態様に係るＣＯＶＩＤ－１９後遺症治療薬のスクリーニング方法で用いるＣＯＶＩＤ－１９後遺症モデル動物は、非ヒト哺乳動物にＳＡＲＳ－ＣｏＶ－２　Ｓ１タンパク質を発現させた動物である。 (COVID-19 sequelae model animal)
A COVID-19 sequelae model animal used in the screening method for a therapeutic drug for COVID-19 sequelae according to one aspect of the present invention is an animal in which SARS-CoV-2 S1 protein is expressed in a non-human mammal.
　ＣＯＶＩＤ－１９後遺症モデル動物において、ＳＡＲＳ－ＣｏＶ－２　Ｓ１タンパク質を一過性に発現させてもよく、恒常的に発現させてもよい。トランスジェニック動物を利用してもよい。発現が終了又は減弱した時点での症状を精査できることから、ＳＡＲＳ－ＣｏＶ－２　Ｓ１タンパク質の発現が一過性であり、本スクリーニング方法の投与工程で使用する時点で、ＣＯＶＩＤ－１９後遺症モデル動物におけるＳＡＲＳ－ＣｏＶ－２　Ｓ１タンパク質の発現が終了又は減弱していることが好ましい。 In COVID-19 sequelae model animals, the SARS-CoV-2 S1 protein may be transiently expressed or permanently expressed. Transgenic animals may also be used. Since it is possible to closely examine the symptoms at the time when the expression is terminated or attenuated, the expression of the SARS-CoV-2 S1 protein is transient, and at the time of use in the administration step of this screening method, in the COVID-19 sequelae model animal Expression of the SARS-CoV-2 S1 protein is preferably terminated or attenuated.
　また、ＣＯＶＩＤ－１９後遺症を効率よく発症させることができることから、ＣＯＶＩＤ－１９後遺症モデル動物は、非ヒト哺乳動物の鼻腔及び鼻腔周囲の少なくとも１つにおいてＳＡＲＳ－ＣｏＶ－２　Ｓ１タンパク質を発現させたモデル動物であることが好ましい。ＣＯＶＩＤ－１９後遺症モデル動物は、試験物質を投与する工程の前に炎症が誘導されてもよい。 In addition, since it is possible to efficiently develop the sequelae of COVID-19, a model animal with the sequelae of COVID-19 is a model in which the SARS-CoV-2 S1 protein is expressed in at least one of the nasal and perinasal cavities of non-human mammals. Animals are preferred. In the COVID-19 sequelae model animal, inflammation may be induced prior to the step of administering the test substance.
　非ヒト哺乳動物にＳＡＲＳ－ＣｏＶ－２　Ｓ１タンパク質を発現させる方法は特に限定されない。例えば、後述するＣＯＶＩＤ－１９後遺症モデル動物の製造方法によって非ヒト哺乳動物にＳＡＲＳ－ＣｏＶ－２　Ｓ１タンパク質を発現させて、ＣＯＶＩＤ－１９後遺症モデル動物を製造することができる。 The method for expressing the SARS-CoV-2 S1 protein in non-human mammals is not particularly limited. For example, a COVID-19 sequelae model animal can be produced by expressing the SARS-CoV-2 S1 protein in a non-human mammal according to the method for producing a COVID-19 sequelae model animal described below.
　（投与工程）
　投与工程は、ＣＯＶＩＤ－１９後遺症モデル動物に、試験物質を投与する工程である。試験物質の投与量及び投与方法は特に限定されない。試験物質中の有効成分の濃度、試験物質の剤形などに応じて、適宜設定することができる。 (Administration step)
The administration step is a step of administering a test substance to a COVID-19 sequelae model animal. The dose and administration method of the test substance are not particularly limited. It can be appropriately set according to the concentration of the active ingredient in the test substance, the dosage form of the test substance, and the like.
　（評価工程）
　評価工程は、ＣＯＶＩＤ－１９後遺症モデル動物における、試験物質投与前後の新型コロナウイルスが関与する症状の変化を評価する工程である。 (Evaluation process)
The evaluation step is a step of evaluating changes in symptoms associated with the novel coronavirus before and after administration of the test substance in the COVID-19 sequelae model animal.
　評価工程において、ＣＯＶＩＤ－１９後遺症モデル動物における新型コロナウイルスが関与する症状の変化を評価する方法は特に限定されない。例えば、評価工程では、ＣＯＶＩＤ－１９後遺症モデル動物の行動量の変化を定量することによって、ＣＯＶＩＤ－１９後遺症モデル動物における新型コロナウイルスが関与する症状の変化を評価することができる。 In the evaluation process, the method of evaluating changes in symptoms related to the new coronavirus in COVID-19 sequelae model animals is not particularly limited. For example, in the evaluation step, it is possible to evaluate changes in symptoms associated with the novel coronavirus in the COVID-19 sequelae model animal by quantifying changes in the amount of behavior of the COVID-19 sequelae model animal.
　ＣＯＶＩＤ－１９後遺症モデル動物の行動量の変化を定量する方法としては、実施例に後述する１０％の重り付き強制水泳試験、尾懸垂試験などの行動実験を試験物質投与前後に行うことによって、試験物質投与前後のＣＯＶＩＤ－１９後遺症モデル動物の行動量の変化を定量することができる。 As a method for quantifying changes in the amount of behavior of a model animal with the sequelae of COVID-19, behavioral experiments such as a 10% weighted forced swim test and a tail suspension test described later in the Examples are performed before and after administration of the test substance. It is possible to quantify the change in the amount of behavior of the COVID-19 sequelae model animal before and after administration of the substance.
　また、例えば、評価工程では、ＣＯＶＩＤ－１９後遺症モデル動物の脳における炎症マーカーの発現量の変化を定量することによって、ＣＯＶＩＤ－１９後遺症モデル動物における新型コロナウイルスが関与する症状の変化を確認してもよい。炎症マーカーの例としては、インターロイキン６（ＩＬ－６）、腫瘍壊死因子（ＴＮＦα）、ケモカインＣＣモチーフリガンド２（ＣＣＬ２）などの炎症マーカーを挙げることができる。 In addition, for example, in the evaluation process, by quantifying changes in the expression levels of inflammatory markers in the brains of COVID-19 sequelae model animals, changes in symptoms associated with the novel coronavirus in COVID-19 sequelae model animals are confirmed. good too. Examples of inflammatory markers include inflammatory markers such as interleukin 6 (IL-6), tumor necrosis factor (TNFα), chemokine CC motif ligand 2 (CCL2).
　ＣＯＶＩＤ－１９後遺症モデル動物の脳における炎症マーカーの発現量の変化を定量する場合、同一個体における試験物質投与前後のＣＯＶＩＤ－１９後遺症モデル動物の脳における炎症マーカーの発現量の変化を定量することはできない。従って、上述した炎症マーカーの発現量の測定を試験物質投与群とプラセボ投与群とで比較することによって、試験物質投与前後のＣＯＶＩＤ－１９後遺症モデル動物の脳における炎症マーカーの発現量の変化を定量することができる。 When quantifying the change in the expression level of inflammatory markers in the brain of a COVID-19 sequelae model animal, it is possible to quantify the change in the expression level of the inflammatory marker in the brain of the COVID-19 sequelae model animal before and after administration of the test substance in the same individual. Can not. Therefore, by comparing the measurement of the expression levels of the inflammatory markers described above between the test substance administration group and the placebo administration group, quantification of changes in the expression levels of inflammatory markers in the brains of COVID-19 sequelae model animals before and after administration of the test substance. can do.
　評価工程では、新型コロナウイルスが関与する症状の変化の評価結果に基づき、試験物質のＣＯＶＩＤ－１９後遺症に対する治療効果又は予防効果を評価すればよい。例えば、評価工程において、ＣＯＶＩＤ－１９後遺症モデル動物の行動量の変化を定量することによってＣＯＶＩＤ－１９後遺症モデル動物における新型コロナウイルスが関与する症状の変化を評価した結果、試験物質投与前後で新型コロナウイルスが関与する疲労又はうつ症状が改善されていれば、試験物質はＣＯＶＩＤ－１９後遺症を治療又は予防する活性を有していると評価し得る。 In the evaluation process, the therapeutic or preventive effect of the test substance on the aftereffects of COVID-19 should be evaluated based on the evaluation results of changes in symptoms related to the new coronavirus. For example, in the evaluation process, as a result of evaluating the change in symptoms related to the new coronavirus in the COVID-19 sequelae model animal by quantifying the change in the amount of behavior of the COVID-19 sequelae model animal, the new coronavirus before and after administration of the test substance A test substance can be assessed as having activity in treating or preventing the sequelae of COVID-19 if the symptoms of virus-related fatigue or depression are ameliorated.
　また、例えば、評価工程において、ＣＯＶＩＤ－１９後遺症モデル動物の脳における炎症マーカーの発現量の変化を定量することによってＣＯＶＩＤ－１９後遺症モデル動物における新型コロナウイルスが関与する症状の変化を評価した結果、試験物質投与前後で脳における炎症マーカーの発現量が有意に低下していれば、試験物質はＣＯＶＩＤ－１９後遺症を治療又は予防する活性を有していると評価し得る。 In addition, for example, in the evaluation process, by quantifying the change in the expression level of inflammatory markers in the brain of the COVID-19 sequelae model animal, as a result of evaluating the change in symptoms related to the new coronavirus in the COVID-19 sequelae model animal, If the expression levels of inflammatory markers in the brain are significantly reduced before and after administration of the test substance, it can be evaluated that the test substance has an activity to treat or prevent the sequelae of COVID-19.
　ＣＯＶＩＤ－１９後遺症モデル動物の行動量の変化を定量することによるスクリーニングと、ＣＯＶＩＤ－１９後遺症モデル動物の脳における炎症マーカーの発現量の変化を定量することによるスクリーニングとは、適宜併用することもできる。 Screening by quantifying the change in the amount of behavior of the COVID-19 sequelae model animal and screening by quantifying the change in the expression level of inflammatory markers in the brain of the COVID-19 sequelae model animal can be used in combination as appropriate. .
　本発明の一態様に係る薬剤のスクリーニング方法では、有効な試験物質の絞り込みを行うこともできる。例えば、後述する実施例６ではＣＯＶＩＤ－１９後遺症の脳でアセチルコリンが不足していることを示した。このため、アセチルコリン受容体作動薬を優先順位の高い候補薬とすることができる。 In the drug screening method according to one aspect of the present invention, effective test substances can also be narrowed down. For example, Example 6, which will be described later, showed that acetylcholine is deficient in the brain affected by COVID-19. Therefore, acetylcholine receptor agonists can be high priority drug candidates.
　さらに、後述する実施例１０ではアセチルコリン受容体作動薬のうち、血液脳関門を通過しないα７ニコチン受容体作動薬であるＰＮＵ２８２９８７の脳室投与が脳の炎症を抑制する作用を持つことを示した。この結果は、今後のスクリーニングにおいて、α７ニコチン受容体作動薬の優先順位を上げるべきであることを示唆している。このことは、血液脳関門を通過しないために現実的な治療薬候補にはならない薬剤でも、脳室内投与などの方法によって、薬剤スクリーニングの方法性を示唆するなどの利用法が可能であることを示している。 Furthermore, in Example 10, which will be described later, it was shown that among acetylcholine receptor agonists, PNU282987, an α7 nicotinic receptor agonist that does not cross the blood-brain barrier, was administered to the brain ventricle and had the effect of suppressing brain inflammation. This result suggests that α7 nicotinic receptor agonists should be prioritized in future screening. This suggests that even drugs that do not pass through the blood-brain barrier and therefore cannot be realistic candidates for therapeutic drugs can be used in methods such as intracerebroventricular administration, suggesting the feasibility of drug screening. showing.
　α７ニコチン受容体作動薬に免疫抑制機能があることは知られていたが、そのメカニズムは明らかではなかった。後述する実施例１１ではα７ニコチン受容体作動薬によって免疫抑制分子ＺＦＰ３６の発現が増加することがそのメカニズムであることを示唆している。この結果は、スクリーニング工程において薬剤の治療効果のメカニズムが明らかになることによって、より良い効果判定法の開発や、薬剤の標的分子の同定が可能であることを示唆している。 Although it was known that α7 nicotinic receptor agonists had an immunosuppressive function, the mechanism was not clear. Example 11, which will be described later, suggests that the mechanism is that the α7 nicotinic receptor agonist increases the expression of the immunosuppressive molecule ZFP36. This result suggests that the clarification of the mechanism of the therapeutic effect of drugs in the screening process will enable the development of better efficacy assessment methods and the identification of drug target molecules.
　＜３．モデル動物の製造方法＞
　（特徴）
　本発明の一態様に係るＣＯＶＩＤ－１９後遺症モデル動物の製造方法は、ＳＡＲＳ－ＣｏＶ－２　Ｓ１タンパク質発現ベクターを用いて当該ＳＡＲＳ－ＣｏＶ－２　Ｓ１タンパク質を非ヒト哺乳動物に発現させる発現工程を含む。これにより、非ヒト哺乳動物にＳＡＲＳ－ＣｏＶ－２　Ｓ１タンパク質を発現させたＣＯＶＩＤ－１９後遺症モデル動物を製造することができる。ＣＯＶＩＤ－１９後遺症モデル動物は、ＣＯＶＩＤ－１９後遺症の治療又は予防方法の開発、特に治療薬や予防薬などの薬剤開発において、実験動物として用いることができる。また、ＣＯＶＩＤ－１９後遺症モデル動物は、ＣＯＶＩＤ－１９後遺症の発症原因の研究において、実験動物として用いることができる。従って、持続可能な開発目標（ＳＤＧｓ）の目標３「すべての人に健康と福祉を」に貢献できる。 <3. Method for producing a model animal>
(feature)
A method for producing a COVID-19 sequelae model animal according to one aspect of the present invention includes an expression step of expressing the SARS-CoV-2 S1 protein in a non-human mammal using a SARS-CoV-2 S1 protein expression vector. . This makes it possible to produce a COVID-19 sequelae model animal in which the SARS-CoV-2 S1 protein is expressed in a non-human mammal. A COVID-19 sequelae model animal can be used as an experimental animal in the development of treatment or prevention methods for COVID-19 sequelae, particularly in the development of drugs such as therapeutic drugs and prophylactic drugs. In addition, COVID-19 sequelae model animals can be used as experimental animals in research on the causes of the onset of COVID-19 sequelae. Therefore, it can contribute to Goal 3 of the Sustainable Development Goals (SDGs) "Good health and well-being for all".
　（モデル動物の種類）
　本発明の一態様に係るＣＯＶＩＤ－１９後遺症モデル動物の製造方法によって製造されるＣＯＶＩＤ－１９後遺症モデル動物は、実験動物として用いることができる非ヒト哺乳動物（ヒト以外の哺乳動物）であれば種類は特に限定されない。従って、発現工程において対象となる非ヒト哺乳動物の種類は特に限定されず、製造するモデル動物の使用目的によって適宜選択することができる。発現工程において対象となる非ヒト哺乳動物の例としては、マウス、ラット、モルモット、イヌ、ウサギ、サル、チンパンジーなどを挙げることができる。 (type of model animal)
The COVID-19 sequelae model animal produced by the method for producing a COVID-19 sequelae model animal according to one aspect of the present invention is a non-human mammal (mammal other than human) that can be used as an experimental animal. is not particularly limited. Therefore, the type of non-human mammal to be used in the expression step is not particularly limited, and can be appropriately selected depending on the intended use of the model animal to be produced. Examples of non-human mammals used in the expression step include mice, rats, guinea pigs, dogs, rabbits, monkeys, and chimpanzees.
　新型コロナウイルス感染動物に関しては、サルなどの大型動物が有効であることが知られているが、治療又は予防方法の開発、特に治療薬や予防薬などの薬剤開発においては、マウスなどの小型動物を用いた動物モデルが必要であると考えられる。このような理由から、ＣＯＶＩＤ－１９後遺症モデル動物は、マウスモデルなどの小型動物モデルであることが好ましい。 Large animals such as monkeys are known to be effective against novel coronavirus-infected animals. An animal model using For this reason, the COVID-19 sequelae model animal is preferably a small animal model such as a mouse model.
　（発現工程）
　発現工程は、ＳＡＲＳ－ＣｏＶ－２　Ｓ１タンパク質発現ベクターを用いて当該ＳＡＲＳ－ＣｏＶ－２　Ｓ１タンパク質を非ヒト哺乳動物に発現させる工程である。ＳＡＲＳ－ＣｏＶ－２　Ｓ１タンパク質発現ベクターを用いて当該ＳＡＲＳ－ＣｏＶ－２　Ｓ１タンパク質を非ヒト哺乳動物に発現させることによって、非ヒト哺乳動物にＣＯＶＩＤ－１９後遺症を発現させることができる。 (Expression step)
The expression step is a step of expressing the SARS-CoV-2 S1 protein in a non-human mammal using a SARS-CoV-2 S1 protein expression vector. By expressing the SARS-CoV-2 S1 protein in non-human mammals using the SARS-CoV-2 S1 protein expression vector, the non-human mammals can develop COVID-19 sequelae.
　ウイルス感染では一般的に免疫反応による炎症性サイトカイン産生が生じ、これが感染急性期での疲労やうつ症状を引き起こすことが知られている。しかし、ＣＯＶＩＤ－１９後遺症では、他のウイルスに見られないほど強い疲労やうつ症状がみられること、及びこれらの症状が後遺症として持続することが知られている。このため、新型コロナウイルスは、感染時に神経障害を与えるような強い活性を有するタンパク質を持つことが予想される。そこで、本発明者らは、タンパク質の同定を行うべく鋭意検討し、その結果、ＳＡＲＳ－ＣｏＶ－２ウイルスのＳｐｉｋｅタンパク質（１２７３アミノ酸、ＧｅｎＢａｎｋアクセッション番号ＹＰ　００９７２４３９０）のＳ１領域が、ＣＯＶＩＤ－１９後遺症の原因タンパク質であることを初めて見出した。 Viral infection generally causes immune response to produce inflammatory cytokines, which is known to cause fatigue and depressive symptoms during the acute phase of infection. However, it is known that the aftereffects of COVID-19 cause severe fatigue and depression that are not seen in other viruses, and that these symptoms persist as aftereffects. Therefore, the new coronavirus is expected to have a protein with strong activity that causes neuropathy upon infection. Therefore, the present inventors have made intensive studies to identify the protein, and as a result, the S1 region of the Spike protein (1273 amino acids, GenBank Accession No. YP 009724390) of the SARS-CoV-2 virus was found to be a sequelae of COVID-19. was found to be the causative protein of
　感染性のＳＡＲＳ－ＣｏＶ－２ウイルスそのものを感染させて作製したモデル動物を扱うためには、Ｐ３施設などの高度な封じ込め施設が要求される。一方で、本発明の一態様に係るＣＯＶＩＤ－１９後遺症モデル動物の製造方法は、ＳＡＲＳ－ＣｏＶ－２のウイルスそのものではなく、ＳＡＲＳ－ＣｏＶ－２　Ｓ１タンパク質発現ベクターを用いることによって、ＣＯＶＩＤ－１９後遺症の原因タンパク質であるＳＡＲＳ－ＣｏＶ－２　Ｓ１タンパク質を非ヒト哺乳動物に発現させる。このため、本発明の一態様に係るＣＯＶＩＤ－１９後遺症モデル動物の製造方法によって製造されたモデル動物は、通常の実験環境において使用することができるため、取扱い性に優れている。 In order to handle model animals created by infecting the infectious SARS-CoV-2 virus itself, advanced containment facilities such as P3 facilities are required. On the other hand, the method for producing a COVID-19 sequelae model animal according to one aspect of the present invention uses the SARS-CoV-2 S1 protein expression vector instead of the SARS-CoV-2 virus itself to produce the COVID-19 sequelae. The SARS-CoV-2 S1 protein, which is the causative protein of SARS-CoV-2, is expressed in non-human mammals. Therefore, a model animal produced by the method for producing a model animal with sequelae of COVID-19 according to one aspect of the present invention can be used in a normal experimental environment, and is easy to handle.
　感染性のＳＡＲＳ－ＣｏＶ－２ウイルスそのものを感染させて作製したモデル動物では、急性感染の病原性と後遺症の病原性をヒトと同程度に生じるとは限らないため、急性症状によるモデル動物の生存率が低いことや、モデルとして十分な後遺症が生じないことが考えられる。このため、本発明の一態様に係るＣＯＶＩＤ－１９後遺症モデル動物の製造方法によって製造されたモデル動物は、効率的かつ確実にＣＯＶＩＤ－１９後遺症のモデル動物が得られる点で優れている。 In model animals prepared by infecting the infectious SARS-CoV-2 virus itself, the pathogenicity of acute infection and the pathogenicity of sequelae do not necessarily occur to the same extent as humans, so the survival of model animals due to acute symptoms It is possible that the rate is low and that sufficient sequelae do not occur as a model. Therefore, the animal model produced by the method for producing a model animal with sequelae of COVID-19 according to one aspect of the present invention is excellent in that the model animal with sequelae of COVID-19 can be obtained efficiently and reliably.
　本明細書において「ＳＡＲＳ－ＣｏＶ－２　Ｓ１タンパク質発現ベクターを用いて当該ＳＡＲＳ－ＣｏＶ－２　Ｓ１タンパク質を非ヒト哺乳動物に発現させる」とは、ＳＡＲＳ－ＣｏＶ－２　Ｓ１タンパク質発現ベクターを用いて、対象の非ヒト哺乳動物の体内でＳＡＲＳ－ＣｏＶ－２　Ｓ１タンパク質を発現させることを意味する。 As used herein, "using the SARS-CoV-2 S1 protein expression vector to express the SARS-CoV-2 S1 protein in a non-human mammal" means using the SARS-CoV-2 S1 protein expression vector to It means expressing the SARS-CoV-2 S1 protein in the non-human mammal of interest.
　対象の非ヒト哺乳動物の体内でＳＡＲＳ－ＣｏＶ－２　Ｓ１タンパク質を発現させる部位は特に限定されないが、ＣＯＶＩＤ－１９後遺症を効率よく発症させることができることから、発現工程では、非ヒト哺乳動物の鼻腔及び鼻腔周囲の少なくとも１つにおいてＳＡＲＳ－ＣｏＶ－２　Ｓ１タンパク質を発現させることが好ましい。非ヒト哺乳動物の鼻腔及び鼻腔周囲の少なくとも１つにおいてＳＡＲＳ－ＣｏＶ－２　Ｓ１タンパク質を発現させるために、例えば、発現工程では、ＳＡＲＳ－ＣｏＶ－２　Ｓ１タンパク質発現ベクターを非ヒト哺乳動物の鼻腔に投与してもよい。 The site where the SARS-CoV-2 S1 protein is expressed in the body of the target non-human mammal is not particularly limited. It is preferred to express the SARS-CoV-2 S1 protein in at least one of the nasal cavity and the perinasal cavity. In order to express the SARS-CoV-2 S1 protein in at least one of the nasal and perinasal cavities of the non-human mammal, for example, in the expression step, the SARS-CoV-2 S1 protein expression vector is introduced into the nasal cavity of the non-human mammal. may be administered.
　ＳＡＲＳ－ＣｏＶ－２　Ｓｐｉｋｅタンパク質の構造を図１に示す。Ｓ１領域は、ＳＡＲＳ－ＣｏＶ－２　Ｓｐｉｋｅタンパク質のアミノ酸配列における第１番目～第６８５番目のアミノ酸からなるアミノ酸配列を有する領域である。Ｓ１領域は、シグナルペプチド配列（ＳＰ）、Ｎ末ドメイン（ＮＴＤ）、及び受容体結合ドメイン（ＲＢＤ）を含む。ＳＡＲＳ－ＣｏＶ－２ウイルスのＳｐｉｋｅタンパク質のＳ１領域を含むポリペプチドを、本明細書では「ＳＡＲＳ－ＣｏＶ－２　Ｓ１タンパク質」又は単に「Ｓ１タンパク質」と称する。 The structure of the SARS-CoV-2 Spike protein is shown in Figure 1. The S1 region is a region having an amino acid sequence consisting of the 1st to 685th amino acids in the amino acid sequence of the SARS-CoV-2 Spike protein. The S1 region contains a signal peptide sequence (SP), an N-terminal domain (NTD) and a receptor binding domain (RBD). A polypeptide comprising the S1 region of the Spike protein of the SARS-CoV-2 virus is referred to herein as "SARS-CoV-2 S1 protein" or simply "S1 protein."
　ＳＡＲＳ－ＣｏＶ－２　Ｓ１タンパク質は、例えば、以下（ａ）又は（ｂ）のポリペプチドであってよい：
　　（ａ）ＧｅｎｅＢａｎｋアクセッション番号ＹＰ＿００９７２４３９０に示されるアミノ酸配列のうち、第１番目～第６８５番目のアミノ酸からなるアミノ酸配列（配列番号１）を有するポリペプチド；
　　（ｂ）ＧｅｎｅＢａｎｋアクセッション番号ＹＰ＿００９７２４３９０に示されるアミノ酸配列のうち、第１番目～第６８５番目のアミノ酸からなるアミノ酸配列（配列番号１）と配列同一性８０％以上のアミノ酸配列からなり、且つ細胞に導入すると細胞内カルシウム濃度を上昇させる活性を有しているポリペプチド。 The SARS-CoV-2 S1 protein may be, for example, a polypeptide of (a) or (b):
(a) A polypeptide having an amino acid sequence (SEQ ID NO: 1) consisting of the 1st to 685th amino acids in the amino acid sequence shown in GeneBank Accession No. YP_009724390;
(B) of the amino acid sequence shown in GeneBank Accession No. YP_009724390, consisting of an amino acid sequence (SEQ ID NO: 1) consisting of the 1st to 685th amino acids and an amino acid sequence with a sequence identity of 80% or more, and in cells A polypeptide having the activity of increasing intracellular calcium concentration upon introduction.
　ＳＡＲＳ－ＣｏＶ－２　Ｓ１タンパク質は、６８５アミノ酸からなる、分子量約７６．７ｋＤａのポリペプチドである。 The SARS-CoV-2 S1 protein is a polypeptide with a molecular weight of about 76.7 kDa, consisting of 685 amino acids.
　ＳＡＲＳ－ＣｏＶ－２　Ｓ１タンパク質は、前記（ｂ）のポリペプチドのように、ＧｅｎｅＢａｎｋアクセッション番号ＹＰ＿００９７２４３９０に示されるアミノ酸配列のうち、第１番目～第６８５番目のアミノ酸からなるアミノ酸配列（配列番号１）と配列同一性８０％以上（８５％以上、９０％以上、９５％以上、９８％以上、９９％以上）のアミノ酸配列からなり、且つ細胞に導入すると細胞内カルシウム濃度を上昇させる活性を有しているポリペプチドであってもよい。本明細書において、アミノ酸配列の同一性パーセントは、遺伝情報処理ソフトウエアＧＥＮＥＴＹＸ　Ｖｅｒ．７（ゼネティックス製）を用いて算出した値である。 The SARS-CoV-2 S1 protein, like the polypeptide (b) above, has an amino acid sequence (SEQ ID NO: 1 ) and an amino acid sequence with a sequence identity of 80% or more (85% or more, 90% or more, 95% or more, 98% or more, 99% or more), and have the activity of increasing intracellular calcium concentration when introduced into cells. It may be a polypeptide having As used herein, percent identity of amino acid sequences is calculated using genetic information processing software GENETYX Ver. 7 (manufactured by Genetics).
　また、前記（ｂ）のポリペプチドは、ＧｅｎｅＢａｎｋアクセッション番号ＹＰ＿００９７２４３９０に示されるアミノ酸配列のうち、第１番目～第６８５番目のアミノ酸からなるアミノ酸配列（配列番号１）において、１００個以下のアミノ酸が置換、欠失、挿入、及び／又は付加されたアミノ酸配列からなり、且つ細胞に導入すると細胞内カルシウム濃度を上昇させる活性を有しているポリペプチドであってもよい。本明細書において「１００個以下のアミノ酸が置換、欠失、挿入、及び／又は付加された」とは、部位特異的突然変異誘発法などの公知の変異ペプチド作製法により１００個以下（９０個以下、８０個以下、７０個以下、６０個以下、５０個以下、４０個以下、３０個以下、２０個以下、１５個以下、１０個以下、７個以下、５個以下、又は２個以下）のアミノ酸が置換、欠失、挿入、及び／又は付加されることを意味する。このように、前記（ｂ）のポリペプチドは、前記（ａ）ポリペプチドの変異体であるといえる。なお、ここでいう「変異」は、主として公知の変異タンパク質作製法により人為的に導入された変異を意味するが、天然に存在する同様の変異タンパク質を単離精製したものであってもよい。 In addition, the polypeptide of (b) has 100 or less amino acids in the amino acid sequence (SEQ ID NO: 1) consisting of the 1st to 685th amino acids in the amino acid sequence shown in GeneBank Accession No. YP_009724390. It may be a polypeptide consisting of a substituted, deleted, inserted and/or added amino acid sequence and having the activity of increasing intracellular calcium concentration when introduced into a cell. As used herein, "substitution, deletion, insertion, and/or addition of 100 or less amino acids" means 100 or less (90 80 or less, 70 or less, 60 or less, 50 or less, 40 or less, 30 or less, 20 or less, 15 or less, 10 or less, 7 or less, 5 or less, or 2 or less ) are substituted, deleted, inserted and/or added. Thus, the polypeptide of (b) can be said to be a variant of the polypeptide of (a). The term "mutation" as used herein mainly means a mutation artificially introduced by a known method for producing a mutant protein, but it may be one obtained by isolating and purifying a naturally occurring similar mutant protein.
　ＳＡＲＳ－ＣｏＶ－２ウイルスの変異株がこれまでに報告されており、スパイクタンパク質に変異を有することが知られている。これまでに報告されている変異株におけるＳＡＲＳ－ＣｏＶ－２　Ｓ１タンパク質の主な変異は以下の通りである。（ｂ）のポリペプチドは、これらの変異を有していることが好ましい。
・ＳＡＲＳ－ＣｏＶ－２　Ｂ．１．１．７系統（いわゆる、「アルファ株」）：ｄｅｌｅｔｉｏｎ６９－７０、ｄｅｌｅｔｉｏｎ１４４－１４５、Ｎ５０１Ｙ、Ａ５７０Ｄ、及びＤ６１４Ｇ、Ｐ６８１Ｈ
・ＳＡＲＳ－ＣｏＶ－２　Ｂ．１．３５１系統（いわゆる、「ベータ株」）：Ｄ８０Ａ、Ｄ２１５Ｇ、　Ｄｅｌｅｔｉｏｎ２４１－２４３、Ｋ４１７Ｎ、Ｅ４８４Ｋ、Ｎ５０１Ｙ、及びＤ６１４Ｇ
・ＳＡＲＳ－ＣｏＶ－２　Ｐ．１系統（いわゆる、「ガンマ株」）：Ｌ１８Ｆ，Ｔ２０Ｎ、Ｐ２６Ｓ、Ｄ１３８Ｙ、Ｒ１９０Ｓ、Ｋ４１７Ｔ、Ｅ４８４Ｋ、Ｎ５０１Ｙ、Ｄ６１４Ｇ、及びＨ６５５Ｙ
・ＳＡＲＳ－ＣｏＶ－２　Ｂ．１．６１７．２系統（いわゆる、「デルタ株」）：Ｔ１９Ｒ、Ｇ１４２Ｄ、Ｅ１５６Ｇ、ｄｅｌｅｔｉｏｎ１５７－１５８、Ｌ４５２Ｒ、Ｔ４８７Ｋ、Ｅ４８４Ｑ、Ｄ６１４Ｇ、及びＰ６８１Ｒ
・ＳＡＲＳ－ＣｏＶ－２　Ｂ．１．１．５２９系統（いわゆる、「オミクロン株」）：Ｇ１４２Ｄ、Ｇ３３９Ｄ、Ｓ３７１Ｌ、Ｓ３７３Ｐ、Ｓ３７５Ｆ、Ｋ４１７Ｎ、Ｎ４４０Ｋ、Ｇ４４６Ｓ、Ｓ４７７Ｎ、Ｔ４７８Ｋ、Ｅ４８４Ａ、Ｑ４９３Ｒ、Ｇ４９６Ｓ、Ｑ４９８Ｒ、Ｎ５０１Ｙ、Ｙ５０５Ｈ、Ｄ６１４Ｇ、Ｈ６５５Ｙ、Ｎ６７９Ｋ、及びＰ６８１Ｈ Mutants of the SARS-CoV-2 virus have been previously reported and are known to have mutations in the spike protein. The major mutations of the SARS-CoV-2 S1 protein in mutant strains reported so far are as follows. The polypeptide of (b) preferably has these mutations.
- SARS-CoV-2 B. 1.1.7 strains (so-called “alpha strains”): deletion69-70, deletion144-145, N501Y, A570D, and D614G, P681H
- SARS-CoV-2 B. 1.351 strains (so-called "beta strains"): D80A, D215G, Deletion241-243, K417N, E484K, N501Y, and D614G
・SARS-CoV-2P. One lineage (so-called "gamma strain"): L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N501Y, D614G, and H655Y
- SARS-CoV-2 B. 1.617.2 strains (so-called "delta strains"): T19R, G142D, E156G, deletion157-158, L452R, T487K, E484Q, D614G, and P681R
- SARS-CoV-2 B. 1.1.529 strains (so-called "Omicron strains"): G142D, G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, D614G, H 655Y , N679K, and P681H
　ＳＡＲＳ－ＣｏＶ－２　Ｓ１タンパク質の変異タンパク質が細胞内カルシウム濃度を上昇させる活性を有していることは、該変異タンパク質を任意の培養細胞内で発現させ、細胞内カルシウム濃度を測定することによって確認することができる。細胞内カルシウム濃度は、後述する実施例に記載の方法によって測定することができる。変異タンパク質発現細胞内のカルシウム濃度が、親細胞と比較して有意に上昇している場合に、変異タンパク質が細胞内カルシウム濃度を上昇させる活性を有していると判断することができる。 The SARS-CoV-2 S1 protein mutant protein has the activity of increasing intracellular calcium concentration, which was confirmed by expressing the mutant protein in arbitrary cultured cells and measuring the intracellular calcium concentration. can do. The intracellular calcium concentration can be measured by the method described in Examples below. When the calcium concentration in the mutant protein-expressing cells is significantly increased compared to the parent cell, it can be determined that the mutant protein has the activity of increasing the intracellular calcium concentration.
　ＳＡＲＳ－ＣｏＶ－２　Ｓ１タンパク質発現ベクターは、公知のプラスミドベクター又はウイルスベクターにＳＡＲＳ－ＣｏＶ－２　Ｓ１タンパク質をコードするポリヌクレオチドを導入して得られる発現ベクターである。 A SARS-CoV-2 S1 protein expression vector is an expression vector obtained by introducing a polynucleotide encoding the SARS-CoV-2 S1 protein into a known plasmid vector or viral vector.
　プラスミドベクター又はウイルスベクターは、ＳＡＲＳ－ＣｏＶ－２　Ｓ１タンパク質を非ヒト哺乳動物の体内で発現可能なものであれば、遺伝子組み換えにおいて通常用いられる公知のベクターを適宜選択することができ、種類は特に限定されない。感染効率が高く、非ヒト哺乳動物への導入操作が簡単であることから、ウイルスベクターを好適に使用することができる。ウイルスベクターの例としては、公知の遺伝子治療、ウイルスワクチンなどの目的で通常使用されるアデノウイルスベクターを特に好適に使用することができる。 As the plasmid vector or viral vector, as long as the SARS-CoV-2 S1 protein can be expressed in the body of a non-human mammal, a known vector commonly used in genetic recombination can be appropriately selected. Not limited. Viral vectors can be preferably used because of their high infection efficiency and the ease of introduction into non-human mammals. As an example of a virus vector, an adenovirus vector that is commonly used for purposes such as known gene therapy and virus vaccines can be particularly preferably used.
　ＳＡＲＳ－ＣｏＶ－２　Ｓ１タンパク質を発現させるためのプロモーターは、ＳＡＲＳ－ＣｏＶ－２が感染する細胞においてｍＲＮＡを発現させることが可能であるものであれば、種類は特に限定されない。ＳＡＲＳ－ＣｏＶ－２　Ｓ１タンパク質をＳＡＲＳ－ＣｏＶ－２の感染時と同程度に産生させられるプロモーターを特に好適に使用することができる。 The type of the promoter for expressing the SARS-CoV-2 S1 protein is not particularly limited as long as it can express mRNA in cells infected with SARS-CoV-2. A promoter that allows SARS-CoV-2 S1 protein to be produced to the same extent as during SARS-CoV-2 infection can be particularly preferably used.
　ＳＡＲＳ－ＣｏＶ－２　Ｓ１タンパク質をコードするポリヌクレオチドとしては、例えば、上述した（ａ）又は（ｂ）のポリペプチドをコードするポリヌクレオチドであってよい。 A polynucleotide encoding the SARS-CoV-2 S1 protein may be, for example, a polynucleotide encoding the polypeptide (a) or (b) described above.
　一例として、（ａ）のポリペプチドをコードするポリヌクレオチドの塩基配列を含むＳＡＲＳ－ＣｏＶ－２　Ｓ１の全ゲノム配列は、ＧｅｎＢａｎｋアクセッション番号ＭＮ９０８９４７に公開されている。（ａ）のポリペプチドをコードするポリヌクレオチドは、ＧｅｎｅＢａｎｋアクセッション番号ＭＮ９０８９４７に示される塩基配列のうち、第２１５６３番目～第２３６１７番目のヌクレオチドからなる塩基配列（配列番号２）を有している。（ａ）のポリペプチドをコードするポリヌクレオチドは、２０５５塩基対（約２ｋｂｐ）のサイズを有している。 As an example, the entire genome sequence of SARS-CoV-2 S1, including the base sequence of the polynucleotide encoding the polypeptide of (a), is published in GenBank Accession No. MN908947. The polynucleotide encoding the polypeptide of (a) has a base sequence (SEQ ID NO: 2) consisting of nucleotides 21563-23617 in the base sequence shown in GeneBank Accession No. MN908947. The polynucleotide encoding the polypeptide of (a) has a size of 2055 base pairs (about 2 kbp).
　ＳＡＲＳ－ＣｏＶ－２　Ｓ１タンパク質をコードするポリヌクレオチドを取得する方法は特に限定されない。例えば、ＰＣＲなどの増幅手段を用いる方法を挙げることができる。例えば、ＳＡＲＳ－ＣｏＶ－２　Ｓｐｉｋｅタンパク質をコードする遺伝子のｃＤＮＡ配列のうち、５’側及び３’側の配列（又はその相補配列）の中からそれぞれプライマーを調製し、これらプライマーを用いてゲノムＤＮＡ（又はｃＤＮＡ）などを鋳型にしてＰＣＲなどを行い、両プライマー間に挟まれるＤＮＡ領域を増幅することで、ＳＡＲＳ－ＣｏＶ－２　Ｓ１タンパク質をコードするポリヌクレオチドを含むＤＮＡ断片を大量に取得できる。遺伝子配列情報をもとにして、ＳＡＲＳ－ＣｏＶ－２　Ｓ１タンパク質をコードするポリヌクレオチドの塩基配列を有するポリヌクレオチドを、公知の化学合成を用いて合成してもよい。塩基配列は、使用する動物のコドン使用頻度に最適化された配列を用いてもよい。 The method for obtaining the polynucleotide encoding the SARS-CoV-2 S1 protein is not particularly limited. For example, a method using amplification means such as PCR can be mentioned. For example, among the cDNA sequences of the gene encoding the SARS-CoV-2 Spike protein, primers are prepared from the 5' and 3' sequences (or their complementary sequences), and these primers are used to (or cDNA) as a template to amplify the DNA region sandwiched between the two primers, a large amount of DNA fragments containing the polynucleotide encoding the SARS-CoV-2 S1 protein can be obtained. Based on the gene sequence information, a polynucleotide having the base sequence of the polynucleotide encoding the SARS-CoV-2 S1 protein may be synthesized using known chemical synthesis. A base sequence optimized for the codon usage of the animal to be used may be used.
　発現工程では、ＣＯＶＩＤ－１９後遺症を発現させるために十分な量のＳＡＲＳ－ＣｏＶ－２　Ｓ１タンパク質を発現させることができるように、対象となる非ヒト哺乳動物の種別及び体重を考慮し、ＳＡＲＳ－ＣｏＶ－２　Ｓ１タンパク質の発現量を調整すればよい。 In the expression step, SARS- The expression level of the CoV-2 S1 protein should be adjusted.
　本発明の一態様に係るＣＯＶＩＤ－１９後遺症モデル動物の製造方法における発現工程では、ＳＡＲＳ－ＣｏＶ－２　Ｓ１タンパク質を非ヒト哺乳動物において一過性に発現させてもよく、恒常的に発現させてもよい。ＣＯＶＩＤ－１９後遺症モデル動物を用いたＣＯＶＩＤ－１９後遺症の研究においては、ＳＡＲＳ－ＣｏＶ－２　Ｓ１タンパク質の発現が終了又は減弱した時点での症状を精査できることが望ましいことから、発現工程では、ＳＡＲＳ－ＣｏＶ－２　Ｓ１タンパク質を非ヒト哺乳動物において一過性に発現させることが好ましい。 In the expression step in the method for producing a COVID-19 sequelae model animal according to one aspect of the present invention, the SARS-CoV-2 S1 protein may be transiently expressed in a non-human mammal, or may be permanently expressed. good too. In the study of COVID-19 sequelae using COVID-19 sequelae model animals, it is desirable to be able to closely examine the symptoms at the time when the expression of SARS-CoV-2 S1 protein is terminated or attenuated. It is preferred to transiently express the CoV-2 S1 protein in non-human mammals.
　（炎症誘導工程）
　本発明の一態様に係るＣＯＶＩＤ－１９後遺症モデル動物の製造方法は、非ヒト哺乳動物において炎症を誘導する炎症誘導工程をさらに含むことが好ましい。ＣＯＶＩＤ－１９後遺症では、ＳＡＲＳ－ＣｏＶ－２　Ｓ１タンパク質がＳＡＲＳ－ＣｏＶ－２ウイルス感染による炎症状態で発現している。そこで、本発明の一態様に係るＣＯＶＩＤ－１９後遺症モデル動物の製造方法は、炎症誘導工程をさらに含むことによって、実際のＳＡＲＳ－ＣｏＶ－２ウイルス感染における状況も加味したモデル動物を作製することができる。 (Inflammation induction step)
The method for producing a COVID-19 sequelae model animal according to one aspect of the present invention preferably further includes an inflammation-inducing step of inducing inflammation in the non-human mammal. In COVID-19 sequelae, the SARS-CoV-2 S1 protein is expressed in inflammatory conditions due to SARS-CoV-2 virus infection. Therefore, the method for producing a COVID-19 sequelae model animal according to one aspect of the present invention further includes an inflammation-inducing step, so that it is possible to produce a model animal that takes into consideration the situation of actual SARS-CoV-2 virus infection. can.
　非ヒト哺乳動物における炎症の誘導方法は特に限定されないが、作製したモデル動物の取扱い性の観点から、ウイルス感染によらずに薬剤などによって炎症を誘導することが好ましい。例えば、大腸菌Ｏ１１１などのグラム陰性細菌に由来するリポポリサッカリド（ＬＰＳ）を腹腔内投与することによって非ヒト哺乳動物における炎症を誘導することができる。ＬＰＳは、マクロファージを活性化する生物活性を有することが知られている。 The method of inducing inflammation in non-human mammals is not particularly limited, but from the viewpoint of the ease of handling of the model animals produced, it is preferable to induce inflammation not by viral infection but by drugs or the like. For example, inflammation can be induced in non-human mammals by intraperitoneal administration of lipopolysaccharides (LPS) derived from Gram-negative bacteria such as E. coli O111. LPS is known to have the biological activity of activating macrophages.
　炎症誘導工程は、発現工程の後に行われる。炎症誘導工程を発現工程の後に行うことによって、ＣＯＶＩＤ－１９後遺症を発現しているモデル動物における炎症誘導効果を観察することができる。炎症誘導工程は、発現工程の前に行われてもよい。 The inflammation induction process is performed after the expression process. By performing the inflammation-inducing step after the expression step, the inflammation-inducing effect can be observed in model animals expressing COVID-19 sequelae. The inflammation-inducing step may precede the expressing step.
　（評価工程）
　本発明の一態様に係るＣＯＶＩＤ－１９後遺症モデル動物の製造方法は、発現工程後の非ヒト哺乳動物におけるＣＯＶＩＤ－１９後遺症の症状の程度を評価する評価工程をさらに含んでいてもよい。 (Evaluation process)
The method for producing a COVID-19 sequelae model animal according to one aspect of the present invention may further include an evaluation step of evaluating the degree of symptoms of COVID-19 sequelae in the non-human mammal after the expression step.
　ＣＯＶＩＤ－１９後遺症の代表的な症状は、疲労及びうつ症状である。このため、行動実験とその定量化によって、疲労又はうつ症状の程度を評価することによって、発現工程後の非ヒト哺乳動物におけるＣＯＶＩＤ－１９後遺症の症状の程度を評価することができる。 The typical symptoms of COVID-19 sequelae are fatigue and depression. Therefore, behavioral experiments and their quantification can assess the severity of symptoms of COVID-19 sequelae in non-human mammals after the onset process by assessing the severity of fatigue or depressive symptoms.
　疲労の程度は、後述する実施例に示す１０％の重り付き強制水泳試験を実施することによって評価することができる。また、うつ症状の程度は、後述する実施例に示す尾懸垂試験を実施することによって評価することができる。 The degree of fatigue can be evaluated by conducting a forced swimming test with a weight of 10% shown in the examples below. Further, the degree of depressive symptoms can be evaluated by conducting a tail suspension test shown in Examples described later.
　評価工程を行うことによって、本発明の一態様に係るＣＯＶＩＤ－１９後遺症モデル動物の製造方法によって製造したモデル動物が、確かにＣＯＶＩＤ－１９後遺症の症状を発現していることを確認することができる。評価工程の結果に応じて、発現工程において発現させるＳＡＲＳ－ＣｏＶ－２　Ｓ１タンパク質の量や、発現ベクターの種類、炎症誘導工程において誘導する炎症の程度などを適宜調整することができる。 By performing the evaluation step, it can be confirmed that the model animal produced by the method for producing a model animal with sequelae of COVID-19 according to one aspect of the present invention does indeed exhibit symptoms of sequelae of COVID-19. . Depending on the results of the evaluation step, the amount of SARS-CoV-2 S1 protein to be expressed in the expression step, the type of expression vector, the degree of inflammation to be induced in the inflammation induction step, and the like can be adjusted as appropriate.
　＜４．ＣＯＶＩＤ－１９後遺症モデル動物作製用キット＞ <4. COVID-19 sequelae model animal production kit>
　本発明の一態様に係るＣＯＶＩＤ－１９後遺症モデル動物作製用キット（以下、単に「キット」）は、非ヒト哺乳動物の細胞内でＳＡＲＳ－ＣｏＶ－２　Ｓ１タンパク質を発現可能な発現ベクターを含む。本発明の一態様に係るキットは、本発明の一態様に係るＣＯＶＩＤ－１９後遺症モデル動物の製造方法において好適に使用することができる。 A COVID-19 sequelae animal model production kit (hereinafter simply "kit") according to one aspect of the present invention includes an expression vector capable of expressing SARS-CoV-2 S1 protein in cells of non-human mammals. A kit according to one aspect of the present invention can be suitably used in a method for producing a model animal for the sequelae of COVID-19 according to one aspect of the present invention.
　本発明において、「キット」とは、特定の材料を内包する容器（例えば、ボトル、プレート、チューブ、ディッシュ等）を備えた包装物が意図される。本発明の一態様に係るキットは、そこに含まれる各々の材料が独立して存在している形態であってもよく、複数の材料が混在している形態（例えば、組成物の形態）であってもよい。キットは、各材料を使用するための指示書を備えていることが好ましい。 In the present invention, a "kit" is intended to be a package that includes a container (eg, bottle, plate, tube, dish, etc.) containing specific materials. The kit according to one aspect of the present invention may be in a form in which each material contained therein exists independently, or in a form in which a plurality of materials are mixed (e.g., in the form of a composition) There may be. Kits preferably include instructions for using each material.
　本発明の一態様に係るキットは、本発明の一態様に係るＣＯＶＩＤ－１９後遺症モデル動物の製造方法を実施するための材料を備えていればよく、非ヒト哺乳動物の細胞内でＳＡＲＳ－ＣｏＶ－２　Ｓ１タンパク質を発現可能な発現ベクター以外の具体的なキットの構成、材料、機器などは、特に限定されるものではない。 The kit according to one aspect of the present invention only needs to include materials for carrying out the method for producing a COVID-19 sequelae model animal according to one aspect of the present invention, and SARS-CoV in the cells of non-human mammals. -2 Except for the expression vector capable of expressing the S1 protein, the specific construction of the kit, materials, equipment, etc. are not particularly limited.
　本発明の一態様に係るキットは、非ヒト哺乳動物の細胞内でＳＡＲＳ－ＣｏＶ－２　Ｓ１タンパク質を発現可能な発現ベクターの他に、炎症誘導工程において使用するためのリポポリサッカリド（ＬＰＳ）をさらに備えていてもよい。 The kit according to one aspect of the present invention contains an expression vector capable of expressing the SARS-CoV-2 S1 protein in non-human mammalian cells, and lipopolysaccharide (LPS) for use in the inflammation-inducing step. You may have more.
　＜５．ＣＯＶＩＤ－１９後遺症モデル動物＞
　本発明の一態様に係るＣＯＶＩＤ－１９後遺症モデル動物の製造方法によって製造されたＣＯＶＩＤ－１９後遺症モデル動物もまた、本発明の範疇に含まれる。本発明の一態様に係るＣＯＶＩＤ－１９後遺症モデル動物は、本発明の一態様に係るＣＯＶＩＤ－１９後遺症モデル動物の製造方法によって製造されるため、Ｐ３施設などの高度な封じ込め施設を必要とせず、通常の実験環境において使用することができるため、取扱い性に優れている。 <5. COVID-19 sequelae model animals>
A COVID-19 sequelae model animal produced by the method for producing a COVID-19 sequelae model animal according to one aspect of the present invention is also included in the scope of the present invention. Since the COVID-19 sequelae model animal according to one aspect of the present invention is produced by the method for producing a COVID-19 sequelae model animal according to one aspect of the present invention, it does not require a high-level containment facility such as a P3 facility, Since it can be used in a normal experimental environment, it is easy to handle.
　＜付記事項＞
　以上のように、本発明の一態様は、以下である。
＜１＞アセチルコリン受容体作動薬を有効成分として含む、新型コロナウイルス感染症後遺症治療薬。
＜２＞前記アセチルコリン受容体作動薬は、脳内のアセチルコリン受容体に作用する中枢性のアセチルコリン受容体作動薬である、＜１＞に記載の新型コロナウイルス感染症後遺症治療薬。
＜３＞前記アセチルコリン受容体作動薬は、ドネペジルである、＜１＞又は＜２＞に記載の新型コロナウイルス感染症後遺症治療薬。
＜４＞前記新型コロナウイルス感染症後遺症は、新型コロナウイルスが関与する疲労である、＜１＞～＜３＞の何れか一つに記載の新型コロナウイルス感染症後遺症治療薬。
＜５＞前記新型コロナウイルス感染症後遺症は、新型コロナウイルスが関与するうつ症状である、＜１＞～＜３＞の何れか一つに記載の新型コロナウイルス感染症後遺症治療薬。
＜６＞前記新型コロナウイルス感染症後遺症は、新型コロナウイルスが関与する嗅覚障害である、＜１＞～＜３＞の何れか一つに記載の新型コロナウイルス感染症後遺症治療薬。
＜７＞前記新型コロナウイルス感染症後遺症は、新型コロナウイルスが関与する記憶障害である、＜１＞～＜３＞の何れか一つに記載の新型コロナウイルス感染症後遺症治療薬。
＜８＞非ヒト哺乳動物にＳＡＲＳ－ＣｏＶ－２　Ｓ１タンパク質を発現させた新型コロナウイルス感染症後遺症モデル動物に、試験物質を投与する投与工程と、
　前記モデル動物における、前記試験物質の投与前後の、新型コロナウイルスが関与する症状の変化を評価する評価工程と、を含む新型コロナウイルス感染症後遺症治療薬のスクリーニング方法。
＜９＞前記モデル動物は、前記非ヒト哺乳動物の鼻腔及び鼻腔周囲の少なくとも１つにおいてＳＡＲＳ－ＣｏＶ－２　Ｓ１タンパク質を発現させたモデル動物である、＜８＞に記載の治療薬のスクリーニング方法。
＜１０＞前記モデル動物は、ＳＡＲＳ－ＣｏＶ－２　Ｓ１タンパク質発現ベクターを用いて当該ＳＡＲＳ－ＣｏＶ－２　Ｓ１タンパク質を前記非ヒト哺乳動物に発現させる発現工程を含む、新型コロナウイルス感染症後遺症モデル動物の製造方法によって製造されたモデル動物である、＜８＞又は＜９＞に記載の治療薬のスクリーニング方法。
＜１１＞前記新型コロナウイルス感染症後遺症モデル動物の製造方法は、前記非ヒト哺乳動物において炎症を誘導する炎症誘導工程をさらに含む、＜１０＞に記載の治療薬のスクリーニング方法。
＜１２＞ＳＡＲＳ－ＣｏＶ－２　Ｓ１タンパク質発現ベクターを用いて当該ＳＡＲＳ－ＣｏＶ－２　Ｓ１タンパク質を非ヒト哺乳動物に発現させる発現工程を含む、新型コロナウイルス感染症後遺症モデル動物の製造方法。
＜１３＞前記発現工程では、前記非ヒト哺乳動物の鼻腔及び鼻腔周囲の少なくとも１つにおいてＳＡＲＳ－ＣｏＶ－２　Ｓ１タンパク質を発現させる、＜１２＞に記載の新型コロナウイルス感染症後遺症モデル動物の製造方法。
＜１４＞前記非ヒト哺乳動物において炎症を誘導する炎症誘導工程をさらに含む、＜１２＞又は＜１３＞に記載の新型コロナウイルス感染症後遺症モデル動物の製造方法。
＜１５＞前記ＳＡＲＳ－ＣｏＶ－２　Ｓ１タンパク質は、以下（ａ）又は（ｂ）のポリペプチドである、＜１２＞～＜１４＞の何れか一つに記載の新型コロナウイルス感染症後遺症モデル動物の製造方法：
　　（ａ）ＧｅｎｅＢａｎｋアクセッション番号ＹＰ＿００９７２４３９０に示されるアミノ酸配列のうち、第１番目～第６８５番目のアミノ酸からなるアミノ酸配列（配列番号１）を有するポリペプチド；
　　（ｂ）ＧｅｎｅＢａｎｋアクセッション番号ＹＰ＿００９７２４３９０に示されるアミノ酸配列のうち、第１番目～第６８５番目のアミノ酸からなるアミノ酸配列（配列番号１）と配列同一性８０％以上のアミノ酸配列からなり、且つ細胞に導入すると細胞内カルシウム濃度を上昇させる活性を有しているポリペプチド。 <Additional notes>
As described above, one aspect of the present invention is as follows.
<1> A therapeutic drug for aftereffects of novel coronavirus infection, containing an acetylcholine receptor agonist as an active ingredient.
<2> The therapeutic drug for aftereffects of novel coronavirus infection according to <1>, wherein the acetylcholine receptor agonist is a central acetylcholine receptor agonist that acts on acetylcholine receptors in the brain.
<3> The therapeutic drug for aftereffects of novel coronavirus infection according to <1> or <2>, wherein the acetylcholine receptor agonist is donepezil.
<4> The novel coronavirus infection sequelae therapeutic drug according to any one of <1> to <3>, wherein the novel coronavirus infection sequelae is fatigue associated with the novel coronavirus infection.
<5> The therapeutic drug for aftereffects of novel coronavirus infection according to any one of <1> to <3>, wherein the aftereffects of novel coronavirus infection are depressive symptoms associated with the novel coronavirus.
<6> The therapeutic drug for aftereffects of novel coronavirus infection according to any one of <1> to <3>, wherein the aftereffects of novel coronavirus infection are olfactory disorders associated with the novel coronavirus.
<7> The therapeutic drug for aftereffects of novel coronavirus infection according to any one of <1> to <3>, wherein the aftereffects of novel coronavirus infection are memory impairment associated with the novel coronavirus.
<8> An administration step of administering a test substance to a novel coronavirus infection sequelae model animal that expresses the SARS-CoV-2 S1 protein in a non-human mammal;
and an evaluation step of evaluating changes in symptoms associated with the novel coronavirus before and after administration of the test substance in the model animal.
<9> The therapeutic drug screening method according to <8>, wherein the model animal is a model animal that expresses the SARS-CoV-2 S1 protein in at least one of the nasal and perinasal cavities of the non-human mammal. .
<10> The model animal includes an expression step of expressing the SARS-CoV-2 S1 protein in the non-human mammal using a SARS-CoV-2 S1 protein expression vector. The therapeutic agent screening method according to <8> or <9>, which is a model animal produced by the production method of <8> or <9>.
<11> The therapeutic drug screening method according to <10>, wherein the method for producing the model animal for sequelae of novel coronavirus infection further includes an inflammation-inducing step of inducing inflammation in the non-human mammal.
<12> A method for producing a novel coronavirus infection sequelae model animal, comprising an expression step of expressing the SARS-CoV-2 S1 protein in a non-human mammal using a SARS-CoV-2 S1 protein expression vector.
<13> Production of the novel coronavirus infection sequelae model animal according to <12>, wherein in the expression step, the SARS-CoV-2 S1 protein is expressed in at least one of the nasal cavity and perinasal cavity of the non-human mammal. Method.
<14> The method for producing an animal model of sequelae of novel coronavirus infection according to <12> or <13>, further comprising an inflammation-inducing step of inducing inflammation in the non-human mammal.
<15> The novel coronavirus infection sequelae model animal according to any one of <12> to <14>, wherein the SARS-CoV-2 S1 protein is a polypeptide of (a) or (b) below. Production method of:
(a) A polypeptide having an amino acid sequence (SEQ ID NO: 1) consisting of the 1st to 685th amino acids in the amino acid sequence shown in GeneBank Accession No. YP_009724390;
(B) of the amino acid sequence shown in GeneBank Accession No. YP_009724390, consisting of an amino acid sequence (SEQ ID NO: 1) consisting of the 1st to 685th amino acids and an amino acid sequence with a sequence identity of 80% or more, and in cells A polypeptide having the activity of increasing intracellular calcium concentration upon introduction.
　＜６．ＣＯＶＩＤ－１９後遺症の治療又は予防方法＞
　本発明の一態様に係るＣＯＶＩＤ－１９後遺症の治療薬を利用したＣＯＶＩＤ－１９後遺症の治療又は予防方法もまた、本発明の範疇に含まれる。 <6. Method for treating or preventing sequelae of COVID-19>
Also included within the scope of the present invention is a method of treating or preventing COVID-19 sequelae using the COVID-19 sequelae therapeutic agent according to one aspect of the present invention.
　すなわち、本発明の一態様に係るＣＯＶＩＤ－１９後遺症の治療又は予防方法は、以下である。
＜１６＞アセチルコリン受容体作動薬を有効成分として含む、ＣＯＶＩＤ－１９後遺症治療薬を被験体（例えば、ヒト、又は、非ヒト動物）に投与する工程を含む、ＣＯＶＩＤ－１９後遺症の治療又は予防方法。
＜１７＞前記アセチルコリン受容体作動薬は、脳内のアセチルコリン受容体に作用する中枢性のアセチルコリン受容体作動薬である、＜１６＞に記載のＣＯＶＩＤ－１９後遺症の治療又は予防方法。
＜１８＞前記アセチルコリン受容体作動薬は、ドネペジルである、＜１６＞又は＜１７＞に記載のＣＯＶＩＤ－１９後遺症の治療又は予防方法。
＜１９＞前記ＣＯＶＩＤ－１９後遺症は、新型コロナウイルスが関与する疲労である、＜１６＞～＜１８＞の何れか一つに記載のＣＯＶＩＤ－１９後遺症の治療又は予防方法。
＜２０＞前記ＣＯＶＩＤ－１９後遺症は、新型コロナウイルスが関与するうつ症状である、＜１６＞～＜１８＞の何れか一つに記載のＣＯＶＩＤ－１９後遺症の治療又は予防方法。
＜２１＞前記ＣＯＶＩＤ－１９後遺症は、新型コロナウイルスが関与する嗅覚障害である、＜１６＞～＜１８＞の何れか一つに記載のＣＯＶＩＤ－１９後遺症の治療又は予防方法。
＜２２＞前記ＣＯＶＩＤ－１９後遺症は、新型コロナウイルスが関与する記憶障害である、＜１６＞～＜１８＞の何れか一つに記載のＣＯＶＩＤ－１９後遺症の治療又は予防方法。 That is, a method for treating or preventing the sequelae of COVID-19 according to one aspect of the present invention is as follows.
<16> A method for treating or preventing the aftereffects of COVID-19, which comprises administering to a subject (e.g., human or non-human animal) a drug for treating the aftereffects of COVID-19, which contains an acetylcholine receptor agonist as an active ingredient. .
<17> The method for treating or preventing the sequelae of COVID-19 according to <16>, wherein the acetylcholine receptor agonist is a central acetylcholine receptor agonist that acts on acetylcholine receptors in the brain.
<18> The method for treating or preventing sequelae of COVID-19 according to <16> or <17>, wherein the acetylcholine receptor agonist is donepezil.
<19> The method for treating or preventing the aftereffects of COVID-19 according to any one of <16> to <18>, wherein the aftereffects of COVID-19 are fatigue associated with the novel coronavirus.
<20> The method for treating or preventing the aftereffects of COVID-19 according to any one of <16> to <18>, wherein the aftereffects of COVID-19 are depressive symptoms associated with the novel coronavirus.
<21> The method for treating or preventing the aftereffects of COVID-19 according to any one of <16> to <18>, wherein the aftereffects of COVID-19 are olfactory disorders associated with the novel coronavirus.
<22> The method for treating or preventing the aftereffects of COVID-19 according to any one of <16> to <18>, wherein the aftereffects of COVID-19 are memory disorders associated with the novel coronavirus.
　ＣＯＶＩＤ－１９後遺症の治療薬の投与対象、投与経路、製剤及び処方等は本発明の一態様に係る治療薬について説明したとおりであるのでここでは繰り返さない。投与が簡単であり、投与対象に対する負担が少ないことから、ＣＯＶＩＤ－１９後遺症の治療薬を経口投与することが好ましい。 The administration target, administration route, formulation, prescription, etc. of the therapeutic drug for the aftereffects of COVID-19 are as described for the therapeutic drug according to one aspect of the present invention, and will not be repeated here. Oral administration of therapeutic agents for the aftereffects of COVID-19 is preferred because administration is simple and less of a burden on the administration subject.
　＜７．その他＞
　本発明の一態様に係るＣＯＶＩＤ－１９後遺症の治療薬の製造のためのアセチルコリン受容体作動薬の使用もまた、本発明の範疇に含まれる。 <7. Others>
Also included within the scope of the present invention is the use of an acetylcholine receptor agonist for the manufacture of a therapeutic drug for COVID-19 sequelae according to one aspect of the present invention.
　すなわち、本発明の一態様に係る使用は、以下である。
＜２３＞ＣＯＶＩＤ－１９後遺症の治療薬の製造のためのアセチルコリン受容体作動薬の使用。
＜２４＞前記アセチルコリン受容体作動薬は、脳内のアセチルコリン受容体に作用する中枢性のアセチルコリン受容体作動薬である、＜２３＞に記載の使用。
＜２５＞前記アセチルコリン受容体作動薬は、ドネペジルである、＜２３＞又は＜２４＞に記載の使用。
＜２６＞前記ＣＯＶＩＤ－１９後遺症は、新型コロナウイルスが関与する疲労である、＜２３＞～＜２５＞の何れか一つに記載の使用。
＜２７＞前記ＣＯＶＩＤ－１９後遺症は、新型コロナウイルスが関与するうつ症状である、＜２３＞～＜２５＞の何れか一つに記載の使用。
＜２８＞前記ＣＯＶＩＤ－１９後遺症は、新型コロナウイルスが関与する嗅覚障害である、＜２３＞～＜２５＞の何れか一つに記載の使用。
＜２９＞前記ＣＯＶＩＤ－１９後遺症は、新型コロナウイルスが関与する記憶障害である、＜２３＞～＜２５＞の何れか一つに記載の使用。 That is, uses according to one aspect of the present invention are as follows.
<23> Use of an acetylcholine receptor agonist for the manufacture of a therapeutic drug for the sequelae of COVID-19.
<24> The use according to <23>, wherein the acetylcholine receptor agonist is a central acetylcholine receptor agonist that acts on acetylcholine receptors in the brain.
<25> The use according to <23> or <24>, wherein the acetylcholine receptor agonist is donepezil.
<26> The use according to any one of <23> to <25>, wherein the COVID-19 sequela is fatigue associated with the novel coronavirus.
<27> The use according to any one of <23> to <25>, wherein the aftereffects of COVID-19 are depressive symptoms associated with the novel coronavirus.
<28> The use according to any one of <23> to <25>, wherein the aftereffect of COVID-19 is an olfactory disorder associated with the novel coronavirus.
<29> The use according to any one of <23> to <25>, wherein the aftereffect of COVID-19 is memory impairment associated with novel coronavirus.
　本発明は上述した各実施形態に限定されるものではなく、請求項に示した範囲で種々の変更が可能であり、異なる実施形態にそれぞれ開示された技術的手段を適宜組み合わせて得られる実施形態についても本発明の技術的範囲に含まれる。 The present invention is not limited to the above-described embodiments, but can be modified in various ways within the scope of the claims, and can be obtained by appropriately combining technical means disclosed in different embodiments. is also included in the technical scope of the present invention.
　〔実施例１〕
　ＳＡＲＳ－ＣｏＶ－２　Ｓ１タンパク質の神経毒性を、細胞内カルシウムの上昇を指標として検討した。 [Example 1]
The neurotoxicity of the SARS-CoV-2 S1 protein was examined using intracellular calcium elevation as an index.
　＜方法＞
　哺乳類細胞用発現ベクターの構築
　哺乳類細胞用発現ベクターｐＦｌａｇ－ＣＭＶ－５ａに、ＳＡＲＳ－ＣｏＶ－２　Ｓｐｉｋｅタンパク質（１２７３アミノ酸、ＧｅｎＢａｎｋアクセッション番号ＹＰ＿００９７２４３９０）のＳ１領域（６８５アミノ酸）をコードするＤＮＡ断片（２０５５塩基対）を組み込み、Ｓ１発現ベクタープラスミド（Ｓ１／ｐＦｌａｇ）を構築した。ＳＡＲＳ－ＣｏＶ－２　Ｓｐｉｋｅタンパク質のＳ１領域（以下、単に「Ｓ１タンパク質」）は、ＧｅｎｅＢａｎｋアクセッション番号ＹＰ＿００９７２４３９０に示されるアミノ酸配列のうち、第１番目～第６８５番目のアミノ酸からなるアミノ酸配列（配列番号１）を有するポリペプチドである。Ｓ１タンパク質をコードするＤＮＡ断片の塩基配列は、ＧｅｎＢａｎｋアクセッション番号ＭＮ９０８９４７に公開されている。 <Method>
Construction of expression vector for mammalian cells DNA fragment (2055) encoding S1 region (685 amino acids) of SARS-CoV-2 Spike protein (1273 amino acids, GenBank Accession No. YP_009724390) in mammalian cell expression vector pFlag-CMV-5a base pairs) to construct the S1 expression vector plasmid (S1/pFlag). The S1 region of the SARS-CoV-2 Spike protein (hereinafter simply "S1 protein") is an amino acid sequence consisting of the 1st to 685th amino acids (SEQ ID NO: 1). The nucleotide sequence of the DNA fragment encoding the S1 protein is published under GenBank Accession No. MN908947.
　さらに、Ｓ１発現アデノウイルスベクターを構築するためにＡｄｅｎｏｖｉｒｕｓ　Ｄｕａｌ　Ｅｘｐｒｅｓｓｉｏｎ　Ｋｉｔ　（ＴａＫａＲａ）を用いた。Ｓ１領域をコードするＤＮＡ断片をコスミドベクターｐＡｘＣＡｗｔｉｔ２に組み込み、ＣＡＧプロモーター制御下でＳ１が発現するアデノウイルスベクター（Ｓ１／Ａｄｖ）を構築した。 Furthermore, the Adenovirus Dual Expression Kit (TaKaRa) was used to construct the S1-expressing adenovirus vector. A DNA fragment encoding the S1 region was incorporated into the cosmid vector pAxCAwtit2 to construct an adenovirus vector (S1/Adv) expressing S1 under the control of the CAG promoter.
　＜細胞内カルシウムの測定＞
　マウス及びヒトの細胞においてＳ１タンパク質を発現させるために、マウス皮膚由来繊維芽細胞株３Ｔ３細胞株及びヒト肺胞基底上皮腺癌細胞Ａ５４９株を用いた。ＣａｌＰｈｏｓ　Ｍａｍｍａｌｉａｎ　Ｔｒａｎｓｆｅｃｔｉｏｎ　Ｋｉｔ（ＴａＫａＲａ）を用いてこれらの細胞にＳ１／ｐＦｌａｇを導入しＳ１タンパク質を一過性発現させた。対照として空のベクタープラスミドｐＦｌａｇ－ＣＭＶ－５ａを用いた。 <Measurement of intracellular calcium>
Mouse skin-derived fibroblast cell line 3T3 cell line and human alveolar basal epithelial adenocarcinoma cell line A549 were used to express S1 protein in mouse and human cells. CalPhos Mammalian Transfection Kit (TaKaRa) was used to introduce S1/pFlag into these cells to transiently express S1 protein. Empty vector plasmid pFlag-CMV-5a was used as a control.
　アデノウイルスベクターの場合は、これらの細胞に直接Ｓ１／Ａｄｖを感染させＳ１タンパク質を一過性に発現させた。対照として空のアデノウイルスベクターを用いた。 In the case of the adenovirus vector, these cells were directly infected with S1/Adv to transiently express the S1 protein. An empty adenoviral vector was used as a control.
　上記手法でＳ１タンパク質を発現させた細胞の細胞内カルシウム濃度を測定するために、Ｃａｌｃｉｕｍ　Ｋｉｔ　ＩＩ　－　Ｆｌｕｏ　４（ＤＯＪＩＮＤＯ）を用いてカルシウムイオンと蛍光プローブとを結合させ、各細胞の蛍光強度をＡｒｒａｙＳｃａｎ　ＸＴ（ＴｈｅｒｍｏＦｉｓｈｅｒ）で測定した。 In order to measure the intracellular calcium concentration of cells expressing S1 protein by the above method, calcium ions and fluorescent probes were bound using Calcium Kit II-Fluo 4 (DOJINDO), and the fluorescence intensity of each cell was measured by ArrayScan. Measured with XT (ThermoFisher).
　＜結果＞
　図１に示したとおり、発現ベクター構築に用いたＳ１タンパク質は、ＧｅｎｅＢａｎｋアクセッション番号ＹＰ＿００９７２４３９０に示されるＳＡＲＳ－ＣｏＶ－２　Ｓｐｉｋｅタンパク質のアミノ酸配列のうち、第１番目～第６８５番目のアミノ酸からなるアミノ酸配列（配列番号１）を有するタンパク質である。 <Results>
As shown in FIG. 1, the S1 protein used for constructing the expression vector is the amino acid sequence of the SARS-CoV-2 Spike protein shown in GeneBank Accession No. YP_009724390. A protein having the sequence (SEQ ID NO: 1).
　Ｓ１タンパク質発現プラスミド又は対照プラスミドを用いて３Ｔ３細胞及びＡ５４９細胞を形質転換し、細胞内カルシウム濃度を測定した。結果を図２に示した。図２のグラフの縦軸に示す「Ｉｎｔｅｎｓｉｔｙ／ａｒｅａ」は、単位面積当たりの蛍光強度を表している。図２のグラフの横軸に示す「対照」は対照プラスミドを形質転換した細胞を表し、「Ｓ１／ｐＦｌａｇ」はＳ１タンパク質発現プラスミドを形質転換した細胞を表している。また、図２中に示す水平のバーは中央値を表している。＊＊＊＊：Ｐ＜０．０００１。 　3T3 cells and A549 cells were transformed with the S1 protein expression plasmid or control plasmid, and the intracellular calcium concentration was measured. The results are shown in FIG. "Intensity/area" shown on the vertical axis of the graph in FIG. 2 represents fluorescence intensity per unit area. "Control" shown on the horizontal axis of the graph in FIG. 2 represents cells transformed with the control plasmid, and "S1/pFlag" represents cells transformed with the S1 protein expression plasmid. Moreover, the horizontal bar shown in FIG. 2 represents the median value. ***: P<0.0001.
　図２に示すとおり、いずれの細胞においてもＳ１タンパク質の発現により細胞内カルシウム濃度が上昇していた。 As shown in Figure 2, the intracellular calcium concentration increased due to the expression of the S1 protein in all cells.
　さらに、Ｓ１タンパク質発現アデノウイルス又は対照アデノウイルスを感染させた３Ｔ３細胞及びＡ５４９細胞の細胞内カルシウム濃度を測定した。結果を図３に示した。図３のグラフの縦軸に示す「Ｉｎｔｅｎｓｉｔｙ／ａｒｅａ」は、単位面積当たりの蛍光強度を表している。図３のグラフの横軸に示す「対照」は対照アデノウイルスを感染させた細胞を表し、「Ｓ１／Ａｄｖ」はＳ１タンパク質発現アデノウイルスを感染させた細胞を表している。また、図３中に示す水平のバーは中央値を表している。＊＊＊＊：Ｐ＜０．０００１。 In addition, the intracellular calcium concentration of 3T3 cells and A549 cells infected with S1 protein-expressing adenovirus or control adenovirus was measured. The results are shown in FIG. "Intensity/area" shown on the vertical axis of the graph in FIG. 3 represents fluorescence intensity per unit area. "Control" indicated on the horizontal axis of the graph in FIG. 3 represents cells infected with the control adenovirus, and "S1/Adv" represents cells infected with the S1 protein-expressing adenovirus. Moreover, the horizontal bar shown in FIG. 3 represents the median value. ***: P<0.0001.
　図３に示すとおり、いずれの細胞においてもＳ１タンパク質の発現により細胞内カルシウム濃度が上昇していた（図３）。 As shown in Figure 3, the intracellular calcium concentration increased due to the expression of the S1 protein in all cells (Figure 3).
　従って、マウス及びヒトの細胞はＳ１タンパク質の発現により細胞内カルシウム濃度が上昇することが示された。つまりＳ１タンパク質が神経細胞で発現した場合、細胞内カルシウム濃度が上昇し、神経細胞死を誘導する可能性が示唆された。 Therefore, it was shown that the intracellular calcium concentration in mouse and human cells increased due to the expression of the S1 protein. In other words, it was suggested that when the S1 protein was expressed in nerve cells, the intracellular calcium concentration increased and the possibility of inducing nerve cell death was suggested.
　〔実施例２〕
　Ｓ１タンパク質発現マウス（Ｓ１マウス）における疲労発現
　＜方法＞
　＜Ｓ１タンパク質発現マウス（Ｓ１マウス）の作製＞
　８～９週齢のＣ５７ＢＬ／６マウスにイソフルランを用いて麻酔をかけ、１×１０９ｉｆｕ／ｍＬのＳ１／Ａｄｖ溶液２５μＬを鼻腔に投与し、自然呼吸で吸引させ（発現工程）、Ｓ１マウス（ＣＯＶＩＤ－１９後遺症モデル動物）を作製した。その後、ホームケージに戻して１週間飼育した。対照として、何も発現しない空のアデノウイルスベクター（ｖｅｃｔｏｒ／Ａｄｖ）を鼻腔投与したマウス（対照マウス）を用いた。 [Example 2]
Fatigue expression in S1 protein-expressing mice (S1 mice) <Method>
<Generation of S1 protein-expressing mouse (S1 mouse)>
An 8- to 9-week-old C57BL/6 mouse was anesthetized with isoflurane, 25 μL of a 1×10 9 ifu/mL S1/Adv solution was administered intranasally, and the mouse was aspirated by spontaneous breathing (expression step). (COVID-19 sequelae model animal) was produced. After that, they were returned to their home cages and reared for one week. As a control, mice to which an empty adenoviral vector (vector/Adv) expressing nothing was intranasally administered (control mice) were used.
　＜疲労行動試験＞
　Ｓ１／Ａｄｖ又はｖｅｃｔｏｒ／Ａｄｖの鼻腔投与から６日目に、疲労行動試験として１０％の重り付き強制水泳試験を行った。方法として、試験当日の午前中にＳ１マウス及び対照マウスの体重を測定し、体重の約１０％となる重りを準備した。その重りをＳ１マウス又は対照マウスの尻尾に固定し、強制水泳試験用の水槽に入れ、鼻先が１０秒間水面下に沈むまでの時間を計測した。 <Fatigue behavior test>
Six days after nasal administration of S1/Adv or vector/Adv, a forced swimming test with a weight of 10% was performed as a fatigue behavior test. As a method, the body weights of S1 mice and control mice were measured in the morning of the day of the test, and a weight of about 10% of the body weight was prepared. The weight was fixed to the tail of S1 mice or control mice, placed in a water tank for forced swimming test, and the time until the nose tip was submerged under water for 10 seconds was measured.
　＜結果＞
　１０％の重り付き強制水泳試験の結果を図４に示した。図４のグラフの縦軸に示す「ｓｗｉｍｍｉｎｇ　ｔｉｍｅ」は、マウスを強制水泳試験用の水槽に入れてから鼻先が１０秒間水面下に沈むまでの時間（秒）を表している。また、図４のグラフの横軸に示す「対照」は対照マウスを表し、「Ｓ１」はＳ１マウスを表している。‡：Ｐ＜０．１。 <Results>
The results of the forced swimming test with 10% weight are shown in FIG. “Swimming time” shown on the vertical axis of the graph in FIG. 4 represents the time (seconds) from when the mouse was put into the water tank for the forced swimming test until the tip of the nose was submerged under the water surface for 10 seconds. "Control" shown on the horizontal axis of the graph in FIG. 4 represents control mice, and "S1" represents S1 mice. ‡: P<0.1.
　対照マウスと比較して、Ｓ１マウスは水泳時間が短い傾向がみられた。これは、対照マウスと比較してＳ１マウスが疲れやすい事を示している。 　Compared to control mice, S1 mice tended to have shorter swimming times. This indicates that S1 mice tire more easily than control mice.
　〔実施例３〕
　Ｓ１マウスにおけるうつ病様行動の発現
　＜方法＞
　Ｓ１マウス及び対照マウスは、実施例２と同様の方法によって作製した。うつ病様行動を確認するために、尾懸垂試験を実施した。方法として、Ｓ１／Ａｄｖ又はｖｅｃｔｏｒ／Ａｄｖの鼻腔投与から６日目のＳ１マウス及び対照マウスの尻尾を固定し、１０分間吊り下げ、その様子を録画し、画像解析ソフトＴａｉｌＳｕｓｐＳｃａｎ（ＣｌｅｖｅｒＳｙｓ　Ｉｎｃ）で解析し、無動時間を計測した。 [Example 3]
Expression of depression-like behavior in S1 mice <Method>
S1 mice and control mice were produced by the same method as in Example 2. A tail suspension test was performed to confirm depression-like behavior. As a method, the tails of S1 mice and control mice on the 6th day after nasal administration of S1 / Adv or vector / Adv were fixed, suspended for 10 minutes, recorded, and analyzed with image analysis software TailSuspScan (CleverSys Inc). and the motionless time was measured.
　＜結果＞
　尾懸垂試験の無動時間をプロットした結果を図５に示した。図５のグラフの横軸に示す「対照」は対照マウスを表し、「Ｓ１」はＳ１マウスを表している。＊：Ｐ＜０．０５。 <Results>
FIG. 5 shows the results of plotting the immobility time of the tail suspension test. "Control" shown on the horizontal axis of the graph in FIG. 5 represents control mice, and "S1" represents S1 mice. *: P<0.05.
　対照マウスと比較して、Ｓ１マウスは有意に無動時間が上昇しており、Ｓ１マウスはうつ病様行動を示す事が明らかになった。 　Compared to control mice, S1 mice had significantly increased immobility time, and it was revealed that S1 mice exhibited depression-like behavior.
　〔実施例４〕
　Ｓ１タンパク質を発現させ、ＬＰＳによって炎症を誘導したマウスにおける嗅球神経の障害
　＜方法＞
　Ｓ１マウス及び対照マウスは、実施例２と同様の方法によって作製した。Ｓ１／Ａｄｖ又はｖｅｃｔｏｒ／Ａｄｖの鼻腔投与から７日目のＳ１マウス及び対照マウスに、５ｍｇ／ｋｇとなるよう大腸菌Ｏ１１１由来リポポリサッカリド（ＭＥＲＣＫ）を腹腔投与し、３０分後及び６０分後に嗅球を採取した。 [Example 4]
Olfactory bulb nerve damage in mice expressing S1 protein and induced inflammation by LPS <Method>
S1 mice and control mice were produced by the same method as in Example 2. Escherichia coli O111-derived lipopolysaccharide (MERCK) was intraperitoneally administered to S1 mice and control mice on day 7 after intranasal administration of S1/Adv or vector/Adv at 5 mg/kg, and olfactory bulbs were detected 30 and 60 minutes after intraperitoneal administration. was taken.
　採取した嗅球を用いて、ＲＮｅａｓｙ　Ｍｉｎｉ　Ｋｉｔ（ＱＩＡＧＥＮ）でＲＮＡを精製した。精製ＲＮＡを鋳型にＰｒｉｍｅＳｃｒｉｐｔ　ＲＴ　ｒｅａｇｅｎｔ　Ｋｉｔ（タカラバイオ）を用いてｃＤＮＡを合成した。合成したｃＤＮＡを用いてカルビンジンの遺伝子発現をＲＴ－ｑＰＣＲ解析した。１８Ｓ　ｒＲＮＡの測定結果を標準化に用いた。 Using the collected olfactory bulb, RNA was purified with the RNeasy Mini Kit (QIAGEN). Using the purified RNA as a template, cDNA was synthesized using PrimeScript RT reagent Kit (Takara Bio). Gene expression of calbindin was analyzed by RT-qPCR using the synthesized cDNA. The measurement results of 18S rRNA were used for normalization.
　＜結果＞
　ＲＴ－ｑＰＣＲ解析の結果を図６に示した。図６のグラフの横軸に示す「対照」は対照マウスを表し、「Ｓ１」はＳ１マウスを表している。‡：Ｐ＜０．１，＊＊：Ｐ＜０．０１。 <Results>
The results of RT-qPCR analysis are shown in FIG. "Control" shown on the horizontal axis of the graph in FIG. 6 represents control mice, and "S1" represents S1 mice. ‡: P<0.1, **: P<0.01.
　ＲＴ－ｑＰＣＲ解析の結果、Ｓ１マウスの嗅球のカルビンジン発現量はＬＰＳ投与後３０分で低下傾向がみられ、ＬＰＳ投与後６０分で有意に低下していた。カルビンジンは、成熟ニューロンのマーカーであることから、従って、末梢の炎症が誘導された場合、Ｓ１マウスの嗅球の成熟ニューロンが障害を受けていると考えられた。 As a result of RT-qPCR analysis, the amount of calbindin expression in the olfactory bulb of S1 mice tended to decrease 30 minutes after LPS administration, and significantly decreased 60 minutes after LPS administration. Since calbindin is a marker of mature neurons, it was thought that mature neurons in the olfactory bulb of S1 mice were damaged when peripheral inflammation was induced.
　〔実施例５〕
　Ｓ１タンパク質を発現させ、ＬＰＳによって炎症を誘導したマウスにおける脳神経の障害
　＜方法＞
　Ｓ１マウス及び対照マウスは、実施例２と同様の方法によって作製した。Ｓ１／Ａｄｖ又はｖｅｃｔｏｒ／Ａｄｖの鼻腔投与から７日目のＳ１マウス及び対照マウスに、５ｍｇ／ｋｇとなるよう大腸菌Ｏ１１１由来リポポリサッカリド（ＭＥＲＣＫ）を腹腔投与し、１５分後に嗅球以外の脳を採取した。 [Example 5]
Cranial nerve damage in mice in which S1 protein was expressed and inflammation was induced by LPS <Method>
S1 mice and control mice were produced by the same method as in Example 2. E. coli O111-derived lipopolysaccharide (MERCK) was intraperitoneally administered to S1 mice and control mice on day 7 after intranasal administration of S1/Adv or vector/Adv, and 5 mg/kg of lipopolysaccharide (MERCK) was administered intraperitoneally. Taken.
　採取した脳を用いて、ＲＮｅａｓｙ　Ｍｉｎｉ　Ｋｉｔ（ＱＩＡＧＥＮ）でＲＮＡを精製した。精製ＲＮＡを鋳型にＰｒｉｍｅＳｃｒｉｐｔ　ＲＴ　ｒｅａｇｅｎｔ　Ｋｉｔ（タカラバイオ）を用いてｃＤＮＡを合成した。合成したｃＤＮＡを用いてカルビンジンの遺伝子発現をＲＴ－ｑＰＣＲ解析した。１８Ｓ　ｒＲＮＡの測定結果を標準化に用いた。 Using the collected brain, RNA was purified with the RNeasy Mini Kit (QIAGEN). Using the purified RNA as a template, cDNA was synthesized using PrimeScript RT reagent Kit (Takara Bio). Gene expression of calbindin was analyzed by RT-qPCR using the synthesized cDNA. The measurement results of 18S rRNA were used for normalization.
　＜結果＞
　ＲＴ－ｑＰＣＲ解析の結果を図７に示した。図７のグラフの横軸に示す「対照」は対照マウスを表し、「Ｓ１」はＳ１マウスを表している。‡：Ｐ＜０．１。 <Results>
The results of RT-qPCR analysis are shown in FIG. "Control" shown on the horizontal axis of the graph in FIG. 7 represents control mice, and "S1" represents S1 mice. ‡: P<0.1.
　ＲＴ－ｑＰＣＲ解析の結果、Ｓ１マウスの脳のカルビンジン発現量はＬＰＳ投与後１５分で低下傾向がみられた。従って、末梢の炎症が誘導された場合、Ｓ１マウスの脳の成熟ニューロンが障害を受けていると考えられた。 As a result of RT-qPCR analysis, the amount of calbindin expression in the brain of S1 mice tended to decrease 15 minutes after LPS administration. Therefore, when peripheral inflammation was induced, mature neurons in the S1 mouse brain were thought to be damaged.
　〔実施例６〕
　Ｓ１タンパク質を発現させたマウスにおけるコリン作動性ニューロンの障害
　＜方法＞
　Ｓ１／Ａｄｖ又はｖｅｃｔｏｒ／Ａｄｖの鼻腔投与から７日目のＳ１マウス及び対照マウスから脳を摘出し、１０％中性ホルマリン溶液で固定した。固定化した脳をパラフィン包埋し、中隔野及びブローカの対角帯が観察できる位置（Ｂｒｅｇｍａ　０．６２ｍｍ）で冠状切片（脳切片）を作製した。作製した脳切片について、脱パラフィン処理後に抗原賦活化処理を行い、Ａｎｔｉ－Ｃｈｏｌｉｎｅ　Ａｃｅｔｙｌｔｒａｎｓｆｅｒａｓｅ　抗体（ａｂｃａｍ社製）で蛍光免疫染色した。 [Example 6]
Impairment of cholinergic neurons in mice expressing S1 protein <Method>
Brains were excised from S1 mice and control mice on day 7 after intranasal administration of S1/Adv or vector/Adv and fixed with a 10% neutral formalin solution. The fixed brain was embedded in paraffin, and a coronal section (brain section) was prepared at a position (Bregma 0.62 mm) where the septal area and Broca's diagonal band can be observed. The prepared brain sections were subjected to antigen retrieval treatment after deparaffinization, and fluorescent immunostaining was performed with an Anti-Choline Acetyltransferase antibody (manufactured by Abcam).
　＜結果＞
　蛍光免疫染色の結果を図８に示した。図８より、Ｓ１マウスにおいて、対照マウスと比して、前脳基底部の中隔野（ＭＳ）及びブローカの対角帯（ＤＢ）のコリンアセチルトランスフェラーゼ（ＣｈＡＴ）陽性細胞であるコリン作動性ニューロンの数が低下していることが示唆された。そこで、ＭＳ／ＤＢ領域におけるＣｈＡＴ陽性細胞数を計測した。結果を図９に示す。図９より、Ｓ１マウスにおいて、ＭＳ／ＤＢ領域におけるＣｈＡＴ陽性細胞数が有意に低下していることが示された。＊＊：Ｐ＜０．０１。 <Results>
FIG. 8 shows the results of fluorescent immunostaining. From FIG. 8, in S1 mice, cholinergic neurons, which are choline acetyltransferase (ChAT)-positive cells, in the basal forebrain septal area (MS) and Broca's diagonal zone (DB) compared to control mice. suggested that the numbers were declining. Therefore, the number of ChAT-positive cells in the MS/DB region was counted. The results are shown in FIG. FIG. 9 showed that the number of ChAT-positive cells in the MS/DB region was significantly reduced in S1 mice. **: P<0.01.
　この結果から、Ｓ１マウスは脳内のコリン作動性ニューロンが障害されていることが示された。従って、Ｓ１マウスは脳内のアセチルコリン量が低下している可能性が示唆された。 These results indicated that cholinergic neurons in the brain of S1 mice were damaged. Therefore, it was suggested that the amount of acetylcholine in the brain of the S1 mouse may be reduced.
　〔実施例７〕
　ドネペジル投与によるＳ１マウスの疲労症状の改善
　＜方法＞
　＜ドネペジルの投与＞
　ドネペジル（富士フイルム和光純薬）は、３２ｍｇ／Ｌとなるように水に溶解し、マウスに飲水投与した。これにより、マウスへのドネペジル投与量は４．０ｍｇ／ｋｇ／ｄａｙとなる。ドネペジル投与量は３．０～５．０ｍｇ／ｋｇ／ｄａｙの文献が多いため、４．０ｍｇ／ｋｇ／ｄａｙを採用した。 [Example 7]
Improvement of fatigue symptoms in S1 mice by administration of donepezil <Method>
<Administration of donepezil>
Donepezil (Fujifilm Wako Pure Chemical Industries, Ltd.) was dissolved in water to a concentration of 32 mg/L, and administered to mice through drinking water. This brings the donepezil dose to mice to 4.0 mg/kg/day. Since there are many literatures on donepezil doses of 3.0 to 5.0 mg/kg/day, 4.0 mg/kg/day was adopted.
　図１０に、ドネペジルの投与スキームを示した。ドネペジル溶液は、Ｓ１／Ａｄｖ又はｖｅｃｔｏｒ／Ａｄｖの鼻腔投与直後からＳ１マウス及び対照マウスに投与され、ドネペジル含有している飲水は２日に１回交換した。なお、以下の実施例においてもドネペジルは同様の方法で投与した。 Figure 10 shows the administration scheme of donepezil. The donepezil solution was administered to S1 and control mice immediately after intranasal administration of S1/Adv or vector/Adv, and the donepezil-containing drinking water was changed once every two days. Donepezil was also administered in the following examples in the same manner.
　＜疲労行動試験＞
　Ｓ１／Ａｄｖ又はｖｅｃｔｏｒ／Ａｄｖの鼻腔投与から６日目に、疲労行動試験として１０％の重り付き強制水泳試験を行った。方法として、試験当日の午前中にＳ１マウス及び対照マウスの体重を測定し、体重の約１０％となる重りを準備した。その重りをＳ１マウス及び対照マウスの尻尾に固定し、強制水泳試験用の水槽に入れ、鼻先が１０秒間水面下に沈むまでの時間を計測した。 <Fatigue behavior test>
Six days after nasal administration of S1/Adv or vector/Adv, a forced swimming test with a weight of 10% was performed as a fatigue behavior test. As a method, the body weights of S1 mice and control mice were measured in the morning of the day of the test, and a weight of about 10% of the body weight was prepared. The weights were fixed to the tails of S1 mice and control mice, placed in a water tank for forced swimming test, and the time it took for the tip of the nose to sink under the water surface for 10 seconds was measured.
　＜結果＞
　１０％の重り付き強制水泳試験の結果を図１１に示した。図１１のグラフの縦軸に示す「ｓｗｉｍｍｉｎｇ　ｔｉｍｅ」は、マウスを強制水泳試験用の水槽に入れてから鼻先が１０秒間水面下に沈むまでの時間（秒）を表している。また、図１１のグラフの横軸に示す「対照」は対照マウスを表し、「Ｓ１」はＳ１マウスを表している。＊：Ｐ＜０．０５。 <Results>
The results of the forced swimming test with 10% weight are shown in FIG. “Swimming time” shown on the vertical axis of the graph in FIG. 11 represents the time (seconds) from when the mouse was put into the water tank for the forced swimming test until the tip of the nose was submerged under the water surface for 10 seconds. "Control" shown on the horizontal axis of the graph in FIG. 11 represents control mice, and "S1" represents S1 mice. *: P<0.05.
　ドネペジルを投与していない群（図１１中の「－」）においては、対照マウスと比較して、Ｓ１マウスは水泳時間が有意に短かった。これに対して、ドネペジルを投与した群（図１１中の「ドネペジル」）においては、対照マウスの水泳時間とＳ１マウスの水泳時間とに差は認められなかった。この結果は、Ｓ１タンパク質発現に起因して発現した疲労様行動をドネペジルが改善することを示すと考えられた。 In the group to which donepezil was not administered ("-" in Fig. 11), the swimming time of S1 mice was significantly shorter than that of control mice. In contrast, in the group to which donepezil was administered (“donepezil” in FIG. 11), no difference was observed between the swimming time of the control mice and the swimming time of the S1 mice. This result was thought to indicate that donepezil ameliorated fatigue-like behavior that was caused by S1 protein expression.
　〔実施例８〕
　ドネペジル投与によるＳ１マウスのうつ病様行動の改善
　＜方法＞
　Ｓ１／Ａｄｖ又はｖｅｃｔｏｒ／Ａｄｖの鼻腔投与後、図１０に示した投与スキームに従ってドネペジルを飲水投与していたＳ１マウス及び対照マウスにおいて、うつ病様行動を確認するために、尾懸垂試験を実施した。方法として、Ｓ１／Ａｄｖ又はｖｅｃｔｏｒ／Ａｄｖの鼻腔投与から６日目のＳ１マウス及び対照マウスの尻尾を固定し、１０分間吊り下げ、その様子を録画し、画像解析ソフトＴａｉｌＳｕｓｐＳｃａｎ（ＣｌｅｖｅｒＳｙｓ　Ｉｎｃ）で解析し、無動時間を計測した。 [Example 8]
Improvement of depression-like behavior in S1 mice by administration of donepezil <Method>
After intranasal administration of S1/Adv or vector/Adv, a tail suspension test was performed to confirm depression-like behavior in S1 mice and control mice receiving donepezil in drinking water according to the administration scheme shown in FIG. . As a method, the tails of S1 mice and control mice on the 6th day after nasal administration of S1 / Adv or vector / Adv were fixed, suspended for 10 minutes, recorded, and analyzed with image analysis software TailSuspScan (CleverSys Inc). and the motionless time was measured.
　＜結果＞
　尾懸垂試験の無動時間をプロットした結果を図１２に示した。図１２のグラフの横軸に示す「対照」は対照マウスを表し、「Ｓ１」はＳ１マウスを表している。＊：Ｐ＜０．０５，＊＊＊：Ｐ＜０．００１。 <Results>
FIG. 12 shows the results of plotting the immobility time of the tail suspension test. "Control" shown on the horizontal axis of the graph in FIG. 12 represents control mice, and "S1" represents S1 mice. *: P<0.05, ***: P<0.001.
　ドネペジルを投与していない群（図１２中の「－」）においては、対照マウスと比較して、Ｓ１マウスは有意に無動時間が上昇した。これに対して、ドネペジルを投与した群（図１２中の「ドネペジル」）においては、対照マウスと比較して、Ｓ１マウスで有意に無動時間が低下することが示された。この結果は、Ｓ１タンパク質発現に起因して発現したうつ病様行動をドネペジルが改善することを示すと考えられた。 In the group to which donepezil was not administered ("-" in Fig. 12), the immobility time was significantly increased in S1 mice compared to control mice. In contrast, in the donepezil-administered group (“donepezil” in FIG. 12), the immobility time was significantly reduced in S1 mice compared to control mice. This result was thought to indicate that donepezil ameliorates depressive-like behavior caused by S1 protein expression.
　〔実施例９〕
　ドネペジル投与によるＳ１マウスの脳の炎症の改善
　＜方法＞
　Ｓ１／Ａｄｖ又はｖｅｃｔｏｒ／Ａｄｖの鼻腔投与後、図１０に示した投与スキームに従ってドネペジルを飲水投与していたＳ１マウス及び対照マウスにおいて、Ｓ１／Ａｄｖ又はｖｅｃｔｏｒ／Ａｄｖの鼻腔投与から７日目に嗅球以外の脳を採取した。採取した脳を用いて、ＲＮｅａｓｙ　Ｍｉｎｉ　Ｋｉｔ（ＱＩＡＧＥＮ）でＲＮＡを精製した。精製ＲＮＡを鋳型にＰｒｉｍｅＳｃｒｉｐｔ　ＲＴ　ｒｅａｇｅｎｔ　Ｋｉｔ（タカラバイオ）を用いてｃＤＮＡを合成した。合成したｃＤＮＡを用いてインターロイキン６（ＩＬ－６）、腫瘍壊死因子（ＴＮＦα）、ケモカインＣＣモチーフリガンド２（ＣＣＬ２）の遺伝子発現をＲＴ－ｑＰＣＲ解析した。１８Ｓ　ｒＲＮＡの測定結果を標準化に用いた。 [Example 9]
Improvement of brain inflammation in S1 mice by administration of donepezil <Method>
After intranasal administration of S1/Adv or vector/Adv, in S1 mice and control mice that were administered donepezil in drinking water according to the administration scheme shown in FIG. Other brains were collected. Using the harvested brain, RNA was purified with RNeasy Mini Kit (QIAGEN). Using the purified RNA as a template, cDNA was synthesized using PrimeScript RT reagent Kit (Takara Bio). Using the synthesized cDNA, gene expressions of interleukin 6 (IL-6), tumor necrosis factor (TNFα), and chemokine CC motif ligand 2 (CCL2) were analyzed by RT-qPCR. 18S rRNA measurements were used for normalization.
　＜結果＞
　ＲＴ－ｑＰＣＲ解析の結果を図１３に示した。図１３のグラフの横軸に示す「対照」は対照マウスを表し、「Ｓ１」はＳ１マウスを表している。＊：Ｐ＜０．０５，‡：Ｐ＜０．１。 <Results>
The results of RT-qPCR analysis are shown in FIG. "Control" shown on the horizontal axis of the graph in FIG. 13 represents control mice, and "S1" represents S1 mice. *: P<0.05, ‡: P<0.1.
　ＲＴ－ｑＰＣＲ解析の結果、ドネペジルを投与していない群（図１３中の「－」）においては、Ｓ１マウスの脳のＩＬ－６発現は有意に上昇しており、それ以外のＴＮＦα及びＣＣＬ２の発現も上昇傾向が認められた。これに対して、ドネペジルを投与した群（図１３中の「ドネペジル」）においては、Ｓ１マウスの脳におけるこれらの遺伝子発現は低下傾向がみられ、対照マウスの脳におけるこれらの遺伝子発現との差が認められなかった。従って、Ｓ１マウスは、Ｓ１タンパク質発現に起因して脳の炎症が誘導され、ドネペジルを投与すると脳の炎症が改善されることが示された。 As a result of RT-qPCR analysis, IL-6 expression in the brain of S1 mice was significantly increased in the group to which donepezil was not administered (“-” in FIG. 13), and other TNFα and CCL2 levels were significantly increased. An increasing trend was also observed in the expression. In contrast, in the group to which donepezil was administered (“donepezil” in FIG. 13), the expression of these genes in the brain of S1 mice tended to decrease, and the expression of these genes in the brain of control mice showed a difference. was not accepted. Thus, it was shown that brain inflammation was induced in S1 mice due to S1 protein expression, and administration of donepezil ameliorated brain inflammation.
　〔実施例１０〕
　ドネペジル以外のアセチルコリン受容体作動薬の効果判定
　＜方法＞
　ドネペジルは脳内のアセチルコリン量を増加させることによってその効果を発揮しているため、さらに効果的な治療薬を開発するためには、アセチルコリン受容体に対してより選択的に作用する薬剤を用いたスクリーニングが必要となる。そこで、Ｓ１マウス及び対照マウスにおいて、Ｓ１／Ａｄｖ又はｖｅｃｔｏｒ／Ａｄｖの鼻腔投与から７日目にα７ニコチン性アセチルコリン受容体特異的な作動薬であるＰＮＵ２８２９８７を４００ｎｍｏｌ/匹となるよう脳室内に投与し、Ｓ１マウスの脳炎症に対する治療効果を検討した。 [Example 10]
Evaluation of the effect of acetylcholine receptor agonists other than donepezil <Method>
Since donepezil exerts its effects by increasing the amount of acetylcholine in the brain, in order to develop more effective therapeutic agents, agents that act more selectively on acetylcholine receptors were used. Screening is required. Therefore, in S1 mice and control mice, PNU282987, an α7 nicotinic acetylcholine receptor-specific agonist, was intracerebroventricularly administered at 400 nmol/mouse on day 7 after intranasal administration of S1/Adv or vector/Adv. , examined the therapeutic effect on brain inflammation in S1 mice.
　ＰＮＵ２８２９８７脳室内投与から1時間後の嗅球以外の脳を採取し、ＲＮｅａｓｙ　Ｍｉｎｉ　Ｋｉｔ（ＱＩＡＧＥＮ）でＲＮＡを精製した。精製ＲＮＡを鋳型にＰｒｉｍｅＳｃｒｉｐｔ　ＲＴ　ｒｅａｇｅｎｔ　Ｋｉｔ（タカラバイオ）を用いてｃＤＮＡを合成した。合成したｃＤＮＡを用いてインターロイキン１β（ＩＬ－１β）、インターロイキン６（ＩＬ－６）の遺伝子発現をＲＴ－ｑＰＣＲ解析した。１８Ｓ　ｒＲＮＡの測定結果を標準化に用いた。 One hour after intracerebroventricular administration of PNU282987, brains other than the olfactory bulb were collected, and RNA was purified using the RNeasy Mini Kit (QIAGEN). Using the purified RNA as a template, cDNA was synthesized using PrimeScript RT reagent Kit (Takara Bio). Gene expressions of interleukin 1β (IL-1β) and interleukin 6 (IL-6) were analyzed by RT-qPCR using the synthesized cDNA. The measurement results of 18S rRNA were used for normalization.
　＜結果＞
　ＲＴ－ｑＰＣＲ解析の結果を図１４に示した。図１４のグラフの横軸に示す「対照」は対照マウスを表し、「Ｓ１」はＳ１マウスを表している。＊＊：Ｐ＜０．０１，‡：Ｐ＜０．１。Ｓ１マウスで観察される、疲労やうつ病の分子機構であると考えられる脳内の炎症性サイトカイン遺伝子（ＩＬ－１β及びＩＬ－６）の発現量の増加が、ＰＮＵ２８２９８７の脳室内投与によって抑制された。この結果は、Ｓ１マウスの脳炎症に対してＰＮＵ２８２９８７及びその誘導体が治療薬の候補となりうることを示唆するとともに、α７ニコチン性アセチルコリン受容体作動薬が治療薬のスクリーニングを行う場合に優先されるべき薬剤であることも示唆すると考えられる。 <Results>
The results of RT-qPCR analysis are shown in FIG. "Control" shown on the horizontal axis of the graph in FIG. 14 represents control mice, and "S1" represents S1 mice. **: P<0.01, ‡: P<0.1. Intraventricular administration of PNU282987 suppressed the increase in expression of inflammatory cytokine genes (IL-1β and IL-6) in the brain, which is thought to be the molecular mechanism of fatigue and depression observed in S1 mice. rice field. This result suggests that PNU282987 and its derivatives can be candidate therapeutic agents for brain inflammation in S1 mice, and should be prioritized when α7 nicotinic acetylcholine receptor agonists are screened for therapeutic agents. It is thought that it also suggests that it is a drug.
　〔実施例１１〕
　アセチルコリン受容体作動薬の作用機構の例
　＜方法＞
　実施例１０でのＰＮＵ２８２９８７による脳炎症の治療効果の分子機構を解明するために、炎症抑制効果を持つ宿主タンパク質であるｚｉｎｃ　ｆｉｎｇｅｒ　ｐｒｏｔｅｉｎ　３６（Ｚｆｐ３６）の遺伝子発現量に対するＳ１とＰＮＵ２８２９８７の影響を検討した。具体的には、実施例１０で合成したｃＤＮＡを用いてＺｆｐ３６の遺伝子発現をＲＴ－ｑＰＣＲ解析した。１８Ｓ　ｒＲＮＡの測定結果を標準化に用いた。 [Example 11]
Examples of Action Mechanisms of Acetylcholine Receptor Agonists <Method>
In order to elucidate the molecular mechanism of the therapeutic effect of PNU282987 on brain inflammation in Example 10, the influence of S1 and PNU282987 on the gene expression level of zinc finger protein 36 (Zfp36), a host protein having an anti-inflammatory effect, was examined. . Specifically, using the cDNA synthesized in Example 10, the gene expression of Zfp36 was analyzed by RT-qPCR. 18S rRNA measurements were used for normalization.
　＜結果＞
　ＲＴ－ｑＰＣＲ解析の結果を図１５に示した。図１５のグラフの横軸に示す「対照」は対照マウスを表し、「Ｓ１」はＳ１マウスを表している。＊：Ｐ＜０．０５，＊＊：Ｐ＜０．０１。この結果、Ｓ１マウスはＺＦＰ３６の低下がみられ、ＰＮＵ２８２９８７投与によってＺＦＰ３６の低下が改善されることが分かった。このことは、Ｓ１マウスによる脳炎症が抗炎症作用をもつＺＦＰ３６の減少によることと、アセチルコリン受容体作動薬、特にα７ニコチン性アセチルコリン受容体作動薬がＺＦＰ３６の発現量回復を介して脳炎症に対する治療効果を発揮することを示唆している。この結果は、ＺＦＰ３６が治療薬の標的となることと、その遺伝子であるＺＦＰ３６の発現量が治療薬スクリーニングのためのバイオマーカーとなることを示すものと考えられる。 <Results>
The results of RT-qPCR analysis are shown in FIG. "Control" shown on the horizontal axis of the graph in FIG. 15 represents control mice, and "S1" represents S1 mice. *: P<0.05, **: P<0.01. As a result, it was found that the S1 mouse showed a decrease in ZFP36, and administration of PNU282987 improved the decrease in ZFP36. This suggests that cerebral inflammation in S1 mice is due to the reduction of ZFP36, which has an anti-inflammatory effect, and that acetylcholine receptor agonists, especially α7 nicotinic acetylcholine receptor agonists, are therapeutic for cerebral inflammation through restoration of ZFP36 expression levels. suggests that it works. This result is considered to indicate that ZFP36 serves as a target for therapeutic drugs and that the expression level of its gene, ZFP36, serves as a biomarker for therapeutic drug screening.
　以上の結果から、Ｓ１タンパク質をマウスに発現させることによってＣＯＶＩＤ－１９後遺症のモデルマウスを作製することができること、ＣＯＶＩＤ－１９後遺症のモデルマウスは脳内のコリン作動性ニューロンが障害されていること、及びＣＯＶＩＤ－１９後遺症のモデルマウスにおけるＣＯＶＩＤ－１９後遺症の症状はドネペジルの投与によって改善されることが示された。また、アセチルコリン受容体作動薬の作用機構の解析結果から、アセチルコリン受容体作動薬の中でも、α７ニコチン性アセチルコリン受容体作動薬が、ＣＯＶＩＤ－１９後遺症治療薬のスクリーニングを行う場合に優先されるべき薬剤であることが示された。 From the above results, it is possible to prepare a mouse model of the sequelae of COVID-19 by expressing the S1 protein in mice, and that the mouse model of the sequelae of COVID-19 has impaired cholinergic neurons in the brain. and COVID-19 sequelae symptoms in model mice of COVID-19 sequelae were shown to be ameliorated by administration of donepezil. In addition, from the results of analysis of the mechanism of action of acetylcholine receptor agonists, among acetylcholine receptor agonists, α7 nicotinic acetylcholine receptor agonists should be prioritized when screening for COVID-19 sequelae treatment drugs. was shown to be
　本発明の一態様に係るＣＯＶＩＤ－１９後遺症治療薬は、新型コロナウイルス感染患者におけるＣＯＶＩＤ－１９後遺症の治療又は予防に寄与することができる。また、本発明の一態様に係るＣＯＶＩＤ－１９後遺症治療薬のスクリーニング方法は、新たなＣＯＶＩＤ－１９後遺症治療薬の薬剤開発に寄与することができる。また、本発明の一態様に係るＣＯＶＩＤ－１９後遺症モデル動物の製造方法によって製造したＣＯＶＩＤ－１９後遺症モデル動物は、医薬品開発やＣＯＶＩＤ－１９後遺症についての基礎研究などに利用することができる。 A drug for treating the aftereffects of COVID-19 according to one aspect of the present invention can contribute to the treatment or prevention of the aftereffects of COVID-19 in patients infected with the novel coronavirus. In addition, the screening method for a drug for treating the aftereffects of COVID-19 according to one aspect of the present invention can contribute to the development of new drugs for treating the aftereffects of COVID-19. In addition, a COVID-19 sequelae model animal produced by the method for producing a COVID-19 sequelae model animal according to one aspect of the present invention can be used for drug development and basic research on COVID-19 sequelae.

Claims (15)

  1. 　アセチルコリン受容体作動薬を有効成分として含む、新型コロナウイルス感染症後遺症治療薬。 A therapeutic drug for the aftereffects of a new coronavirus infection that contains an acetylcholine receptor agonist as an active ingredient.
  2. 　前記アセチルコリン受容体作動薬は、脳内のアセチルコリン受容体に作用する中枢性のアセチルコリン受容体作動薬である、請求項１に記載の新型コロナウイルス感染症後遺症治療薬。 The novel coronavirus infection sequela therapeutic drug according to claim 1, wherein the acetylcholine receptor agonist is a central acetylcholine receptor agonist that acts on acetylcholine receptors in the brain.
  3. 　前記アセチルコリン受容体作動薬は、ドネペジルである、請求項１に記載の新型コロナウイルス感染症後遺症治療薬。 The drug for treating sequelae of novel coronavirus infection according to claim 1, wherein the acetylcholine receptor agonist is donepezil.
  4. 　前記新型コロナウイルス感染症後遺症は、新型コロナウイルスが関与する疲労である、請求項１～３の何れか一項に記載の新型コロナウイルス感染症後遺症治療薬。 The therapeutic drug for aftereffects of novel coronavirus infection according to any one of claims 1 to 3, wherein the aftereffects of novel coronavirus infection are fatigue associated with the novel coronavirus.
  5. 　前記新型コロナウイルス感染症後遺症は、新型コロナウイルスが関与するうつ症状である、請求項１～３の何れか一項に記載の新型コロナウイルス感染症後遺症治療薬。 The drug for treating the aftereffects of the novel coronavirus infection according to any one of claims 1 to 3, wherein the aftereffects of the new coronavirus infection are depressive symptoms associated with the new coronavirus infection.
  6. 　前記新型コロナウイルス感染症後遺症は、新型コロナウイルスが関与する嗅覚障害である、請求項１～３の何れか一項に記載の新型コロナウイルス感染症後遺症治療薬。 The therapeutic drug for the aftereffects of the novel coronavirus infection according to any one of claims 1 to 3, wherein the aftereffects of the new coronavirus infection are olfactory disorders associated with the new coronavirus infection.
  7. 　前記新型コロナウイルス感染症後遺症は、新型コロナウイルスが関与する記憶障害である、請求項１～３の何れか一項に記載の新型コロナウイルス感染症後遺症治療薬。 The therapeutic drug for aftereffects of novel coronavirus infection according to any one of claims 1 to 3, wherein the aftereffects of novel coronavirus infection are memory impairment associated with the novel coronavirus.
  8. 　非ヒト哺乳動物にＳＡＲＳ－ＣｏＶ－２　Ｓ１タンパク質を発現させた新型コロナウイルス感染症後遺症モデル動物に、試験物質を投与する投与工程と、
    　前記モデル動物における、前記試験物質の投与前後の、新型コロナウイルスが関与する症状の変化を評価する評価工程と、を含む新型コロナウイルス感染症後遺症治療薬のスクリーニング方法。     An administration step of administering a test substance to a novel coronavirus infection sequelae model animal in which SARS-CoV-2 S1 protein is expressed in a non-human mammal;
    and an evaluation step of evaluating changes in symptoms associated with the novel coronavirus before and after administration of the test substance in the model animal.
  9. 　前記モデル動物は、前記非ヒト哺乳動物の鼻腔及び鼻腔周囲の少なくとも１つにおいてＳＡＲＳ－ＣｏＶ－２　Ｓ１タンパク質を発現させたモデル動物である、請求項８に記載の治療薬のスクリーニング方法。 The therapeutic agent screening method according to claim 8, wherein the model animal is a model animal in which the SARS-CoV-2 S1 protein is expressed in at least one of the nasal cavity and perinasal cavity of the non-human mammal.
  10. 　前記モデル動物は、ＳＡＲＳ－ＣｏＶ－２　Ｓ１タンパク質発現ベクターを用いて当該ＳＡＲＳ－ＣｏＶ－２　Ｓ１タンパク質を前記非ヒト哺乳動物に発現させる発現工程を含む、新型コロナウイルス感染症後遺症モデル動物の製造方法によって製造されたモデル動物である、請求項８又は９に記載の治療薬のスクリーニング方法。 A method for producing a novel coronavirus infection sequelae model animal, wherein the model animal includes an expression step of expressing the SARS-CoV-2 S1 protein in the non-human mammal using a SARS-CoV-2 S1 protein expression vector. The method for screening a therapeutic agent according to claim 8 or 9, which is a model animal produced by
  11. 　前記新型コロナウイルス感染症後遺症モデル動物の製造方法は、前記非ヒト哺乳動物において炎症を誘導する炎症誘導工程をさらに含む、請求項１０に記載の治療薬のスクリーニング方法。 The therapeutic agent screening method according to claim 10, wherein the method for producing the novel coronavirus infection sequelae model animal further includes an inflammation-inducing step of inducing inflammation in the non-human mammal.
  12. 　ＳＡＲＳ－ＣｏＶ－２　Ｓ１タンパク質発現ベクターを用いて当該ＳＡＲＳ－ＣｏＶ－２　Ｓ１タンパク質を非ヒト哺乳動物に発現させる発現工程を含む、新型コロナウイルス感染症後遺症モデル動物の製造方法。 A method for producing a novel coronavirus infection sequelae model animal, comprising an expression step of expressing the SARS-CoV-2 S1 protein in a non-human mammal using a SARS-CoV-2 S1 protein expression vector.
  13. 　前記発現工程では、前記非ヒト哺乳動物の鼻腔及び鼻腔周囲の少なくとも１つにおいてＳＡＲＳ－ＣｏＶ－２　Ｓ１タンパク質を発現させる、請求項１２に記載の新型コロナウイルス感染症後遺症モデル動物の製造方法。 The method for producing an animal model of sequelae of novel coronavirus infection according to claim 12, wherein, in the expression step, the SARS-CoV-2 S1 protein is expressed in at least one of the nasal cavity and perinasal cavity of the non-human mammal.
  14. 　前記非ヒト哺乳動物において炎症を誘導する炎症誘導工程をさらに含む、請求項１２に記載の新型コロナウイルス感染症後遺症モデル動物の製造方法。 The method for producing a novel coronavirus infection sequelae model animal according to claim 12, further comprising an inflammation-inducing step of inducing inflammation in the non-human mammal.
  15. 　前記ＳＡＲＳ－ＣｏＶ－２　Ｓ１タンパク質は、以下（ａ）又は（ｂ）のポリペプチドである、請求項１２～１４の何れか一項に記載の新型コロナウイルス感染症後遺症モデル動物の製造方法：
    　　（ａ）配列番号１に示されるアミノ酸配列を有するポリペプチド；
    　　（ｂ）配列番号１に示されるアミノ酸配列と配列同一性８０％以上のアミノ酸配列からなり、且つ細胞に導入すると細胞内カルシウム濃度を上昇させる活性を有しているポリペプチド。     The method for producing a novel coronavirus infection sequelae model animal according to any one of claims 12 to 14, wherein the SARS-CoV-2 S1 protein is the following polypeptide (a) or (b):
    (a) a polypeptide having the amino acid sequence shown in SEQ ID NO: 1;
    (b) A polypeptide consisting of an amino acid sequence having a sequence identity of 80% or more with the amino acid sequence shown in SEQ ID NO: 1, and having an activity of increasing intracellular calcium concentration when introduced into a cell.
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